Maximum intensity projections of expression patterns seen in a set of GAL4 lines. (A) A line with no brain expression. (B–I) Lines showing expression in the central complex sorted by increasing number of overall stained cell bodies. GAL4-driven GFP expression is shown in green. The nc82 reference stain, displayed at one-third intensity to prevent obstruction of the GFP signal, is shown in magenta. Line names are given in the lower left of each panel and the estimated number of cells expressing GFP in the central brain is shown in the lower right. For R39H01, shown in (F), 2,000 of the 3,000 estimated cells are mushroom body Kenyon cells. The scale bar shown in (I) is 100 μm. (J–L) are individual optical sections from the specimens above (G–I) taken at the level of the frontal ellipsoid body. Note that, even in lines of the cell density shown in (J) and (K), fine details of the expression pattern can be seen.

Maximum intensity projections of a selected set of sparsely expressing GAL4 lines. GAL4-driven GFP expression is shown in green. The nc82 reference stain, displayed at one-third intensity to prevent obstruction of the GFP signal, is shown in magenta. In (A–F) only the central brain and optic lobes are shown. In (G–I), we also show the VNCs. Line names are given in the lower left of each panel. The scale bar shown in (I) is 100 μm. Abbreviations: ABD, abdominal ganglia; AL, antennal lobe; BU, lateral bulb (lateral triangle); EB, ellipsoid body; FB, fan-shaped body; GA, gall; LA, lamina; LAL, lateral accessory lobe; MB, mushroom body; ME, medulla; NO, noduli; OF, optic focus; OL, optic lobe; PB, protocerebral bridge; PRW, prow; T1, thoracic ganglia 1; T2, thoracic ganglia 2; T3, thoracic ganglia 3; VLP, ventrolateral protocerebrum; wing, wing neuropil; and WED, wedge.

Examples of antennal lobe projection neurons. (A, B, E, F, I and J) Maximum intensity projections of GAL4-driven GFP expression patterns; (A, E and I) include the reference staining and (B, F, and J) show only the GFP signal. (C, G, and K) show color-enhanced direct volume renderings of the signal channel where the signal of interest is false-colored in green and the rest of the pattern is in grey. (D, H and L) Surface reconstructions of the signals of interest in relation to a surface model of the brain. The images in panels (C, D, G, H, K and L) were generated using Amira from the original confocal stacks by segmentation of the neurons of interest using local thresholding, followed by direct volume rendering or surface reconstruction. R14A02 (A–D) and R17G06 (E–H) show examples of the two axon tracts used by most projection neurons, the medial antennal lobe tract (mALT) and the medio-lateral antennal lobe tract (mlALT), respectively. Both lines express in central antennal lobe (AL) glomeruli (R14A02: DL1, D1DA4m, VA7m, VA6; R17G06: DL1, DL5). In R14A02, the axons follow the mALT to the mushroom body (MB) calyx and the lateral horn (LH), while in R17G06 they reach the LH via the mlALT. These patterns are displayed with increased transparency in (D, H, L, M, N and O) where they serve to indicate the positions of the mALT (orange) and the mlALT (purple). R60H12 (I–L) shows a novel population of transverse antennal lobe projection neurons (ALtPN). The cell bodies of these neurons are located ventrally to the antennal lobe (AL) and project into the basal VP3 glomerulus establishing very dense arborizations in the dorso-lateral part of this region. From there they follow the path of the mlALT until they reach the level of the fan-shaped body, where they turn medially, corkscrew halfway around the MB peduncle and arborize densely in a very confined, bar shaped area of the anterior dorso-lateral MB calyx. This region appears not to be the accessory calyx, which is described to be located more medial. Therefore we suggest naming this area lateral accessory calyx (lACA). Earlier descriptions of ALtPNs (; ; ) show a certain similarity to these neurons but the detailed morphology of the cells in R60H12 differ from those in earlier descriptions. Following the nomenclature of we suggest calling these cells AL-t5PN1. In addition to these neurons, a subpopulation in this expression pattern projects along the mlALT to the LH. With the original GAL4 pattern, it is not clear if they are a different population or if the same neurons also bifurcate to the MB calyx and the LH. However, the difference in the diameter of the proximal tracts suggests this expression pattern consists two different cell types. (M–O) 3-D surface renderings from three different angles to display the morphologies of the neurons shown in L. The scale bars in (K) and (L) are 100 μm.

Brain asymmetry revealed by a neuronal population in line R72A10. (A) A 3-D surface reconstruction showing a superposition of this neuronal population in three brains; the same false colors are used in all panels. (B) Sagittal section (3-D direct volume, cut surface rendering) though the central complex showing the three color-coded specimens from (A) at the level of the AB. The dashed orange line indicates the center of the volume projected in (C) and (D). (C) A maximum intensity projection of a 65-μm substack through the FB at the level of the AB showing the tracts from one specimen (green in A, B and D) innervating the AB. The nc82 reference pattern, displayed at two-thirds intensity, is shown in magenta; GFP expression is in green. (D) A maximum intensity projection of 10μm of the three specimens shown in (A), illustrating the position of the AB relative to the FB and NO; the nc82 reference stain is shown in grey. Note how the aborizations of the neurons from the three specimens, shown in different false colors, heavily overlap. The dashed orange line in (C) and (D) indicates section plane shown in (B). AB, asymmetric body; BU, lateral bulb; FB, fan-shaped body; NO, noduli; PB, protocerebral bridge; SLP, superior lateral protocerebrum.

3-D surface reconstructions of 6 specimens of line R26B04 illustrating variability of the same small cell population between individual flies. (A) While establishing the same overall connectivity between the DL3 glomerulus of the antennal lobe (AL), the mushroom body calyx (CA) and the lateral horn (LH), the cells in this population (7±1 cells) show extensive variability in fine morphology and cell body cluster (CBC) position when the brains of different animals are compared. Neurons from different animals are shown in different false colors. (B) – (G) The area in the dashed box in (A) is shown separately for each specimen. The arrows indicate the position of the CBC.

Examples of the numerical coding of expression intensity and distribution scales in the fan-shaped body from different GAL4 lines. These maximum intensity projections of isolated FB volumes display false-color maps generated to facilitate the proofreading process. A false-color scale bar is shown.

The 68 major brain regions shown in false color. See Ito et al. (submitted) for details. Abbreviations for those brain regions visible in this view: AL, antennal lobe; AME, accessory medulla; AMMC, antenna-mechanosensory and motor center; CAN, cantle; CRE, crepine; EB, ellipsoid body; FB, fan-shaped body; LH, lateral horn; LO, lobula; LOP, lobula plate; MB, mushroom body; ME, medulla; NO, noduli; OTU, optic tubercle; PRW, prow; SAD, saddle; SLP, superior lateral protocerebrum; SMP, superior medial protocerebrum; SEG, subesophageal ganglion; VLP, ventrolateral protocerebrum; WED, wedge.