Article Figures & Data
Figures
- Figure 1.
Position and internal organization of the AOTu in osmium-stained sections. A, Overview (frontal section) of the anteriormost part of the bee brain showing the position of the right (B) and left (C–E) AOTus. Boxed areas enlarged in B and C–E, respectively. B, Enlargement revealing the internal compartmentalization of the right AOTu. C–E, Frontal sections of the left AOTu at different depths from the anterior brain surface (C, 30 μm; D, 60 μm; E, 80 μm). F, Vertical section of the left brain hemisphere; boxed area enlarged in G. G, Detail of the AOTu vertically sectioned through the center of the MU and revealing the posteriorly positioned LU. AL, Antennal lobe; MBvl, mushroom-body vertical lobe; MBca, mushroom-body calyx; Me, medulla; Lo, lobula; med, medial; do, dorsal; ant, anterior; lat, lateral. Scale bars, 50 μm.
- Figure 2.
Three-dimensional structure and neural connectivity of the AOTu. A, Three-dimensional reconstruction of confocal image stacks (anterior, posterior, ventral, and dorsal views) showing the different AOTu compartments: MU-DL, green; MU-VL, yellow; VLU, red; LU, blue. This example shows a left AOTu. B, Schematic diagram summarizing neural pathways connecting the AOTu with other brain neuropils. La, Lamina; Me, medulla; Lo, lobulla; AL, antennal lobe; MBvl, mushroom-body vertical lobe; MBca, mushroom-body calyx; CB, central body; LAL, lateral accessory lobe.
- Figure 3.
Segregated input from the optic lobe to the AOTu. A, Dextran tracer injection into the AOTu (as in Fig. 4A) reveals visual input from the medulla (Me) and lobula (Lo) via the AOT. Transmedullary neurons have cell bodies situated along the distal face of the medulla, traverse the medulla from distal to proximal featuring putative input regions in the outer medulla and usually short tangential diversions from their centripetal course within the serpentine (Se) layer (B, C), and are bundled in many fascicles on reaching the inner chiasm. Red arrow in A indicates fibers originating from the dorsalmost part of the medulla and entering the dorsalmost part of the lobula; boxed areas represent comparable regions from different preparations detailed in B, C, and F, respectively. D, Simultaneous injections of two tracers with different colors into the dorsal (red) and ventral (green) medulla show that projections from the medulla are segregated into different AOTu regions; boxed area enlarged in E. Lobula neurons sending input to the AOTu (A) are mainly columnar neurons with dendrites in proximal layers 1–4 (F, G). H, Dorsoventral segregation of input to the AOTu was also identified when tracer injections were simultaneously performed in the ventral (in red) and dorsal (in green) lobula. Boxed area enlarged in I; abbreviations are as defined in Figures 1 and 2.
- Figure 4.
Two separate inter-tubercle tracts connecting the AOTs of both brain hemispheres. A, Composite of frontal sections after tracer injection into the AOTu showing visual input from the optic lobe (see details in Fig. 3) and inter-tubercle connection. The thick arrow indicates input fibers originating from the dorsalmost part of the medulla. The vITT runs ventrally in the anterior surface of the brain, whereas the mITT runs laterally together with the TALT. B, Superposition of frontal protocerebral sections showing vITT and mITT stained after tracer injection into the left AOTu. The mITT is posterior in relation to the vITT and projects dorsally through the protocerebrum, making a loop around the vertical lobes of the ipsilateral and contralateral mushroom bodies (MBvl) before projecting ventrally again to terminate in the contralateral AOTu. C, mITT neurons possess very thin collaterals in the central brain (thick arrow in inset). D, E, Arborizations of inter-tubercle neurons in distinct AOTu compartments. Frontal section of the AOTu anterior (D) and posterior (E) side after tracer injection in the contralateral AOTu. All four AOTu compartments are filled with arborizations of inter-tubercle neurons. Whereas the vITT enters the MU anteriorly, the mITT enters the AOTu dorsolaterally at the inner, more posterior face of the MU. F, An interlobulae neuron previously described as horizontal regressive motion sensitive (HR-n) by DeVoe et al. (1982) runs in very close proximity to the vITT trajectory. The large HR-n bypasses the posterior medial side of the AOTu and possesses a large cell body (asterisk) situated ventrolaterally to the AOTu. However, it does not seem to be associated or connected with the AOTu but exclusively interconnects the lobulae (Lo) of both brain hemispheres, in which it presents wide arborizations. Abbreviations are as defined in Figures 1 and 2.
- Figure 5.
A mushroom-body extrinsic neuron that terminates in the AOTu. A, Dextran-coupled dye injection into the AOTu typically labels the A5-2 neuron (Rybak and Menzel, 1993) with cell body situated close to the inner side of the contralateral MBvl. This large neuron presents a very thick dendritic arbor in the ventral part of the ipsilateral MBvl (B). C, Dextran-coupled dye injection into the ventral part of the mushroom-body vertical lobe labels a network of fine A5-2 axon collaterals in the AOTu. Areas boxed in A and C, respectively, are enlarged in B and D, respectively. D, Thin axons originating from the A5-2 neuron penetrate the inner side of the AOTu and send two main axonal branches into the MU-DL and MU-VL that end in numerous blebby varicosities. Abbreviations are as defined in Figures 1 and 2. Scale bars, 100 μm.
- Figure 6.
AOTu output to the median protocerebrum through the TALT. A, Localized tracer injection into the LAL reveals neurons of the TALT with dendritic ramifications in the AOTu, as well as neurons connecting the LAL with the lower and upper units of the central complex (CB-UU and CB-LU). Fibers of the TALT run laterally and turn around the ipsilateral MBvl before entering the LAL. B, C, Frontal sections of the AOTu showing different types of TALT neurons with arborizations in the AOTu. B, TALT fibers whose dendrites form a dense network of arborizations in the MU-VL and in the LU. C, A composite of all the confocal sections from the AOTu stained in A shows a large number of labeled TALT fibers, including fibers that present conspicuous thick branches on the posteriormost side of the MU and the LU. Abbreviations are as defined in Figures 1 and 2. Scale bars, 50 μm.
- Figure 7.
Dextran injections into the AOTu revealing conspicuous types of TALT axon terminals in the LAL. A, AOTu output neurons of the TALT widely arborizing the ventral LAL. B, High concentration of microglomerular terminals forming a grape-like cluster ventrolaterally with respect to the lower unit of the central complex (CB-LU). C, Another set of neurons also presenting such large microglomerular terminals in the vicinity of the CB. D, E, Enlargements of the microglomerular clusters in preparations B and C, respectively. Although supplied by very thin axon collaterals, these microglomerular terminals are as large as small cell bodies: up to 4 μm wide and 6 μm long. Abbreviations are as defined in Figures 1 and 2. CB-UU, Upper unit of the central complex.
- Figure 8.
Optical recordings of calcium signals from inter-tubercle neurons in the honeybee AOTu. A, Bees are placed individually in opaque recording chambers. The brain area is optically isolated from the compound eye area in which visual stimuli are given, using opaque barriers glued to the bee head with custom black wax. B, Visual stimulation is produced by three rectangular LED arrays (3 × 5 LEDs each) disposed in a half-circle to stimulate different parts of the visual field (dorsal, lateral, or ventral) of the bee right eye. The center of each array is placed at a distance of 6 cm from the eye, corresponding to an angular subtense of 24.5° (anteroposterior axis) and 17.1° (dorsoventral axis) at the bee eye. C, False color-coded activation maps (percentage change in 340 nm/380 nm fluorescence ratio; see Materials and Methods) obtained in the right AOTu for two different animals (bee 1 and bee 2). The contours of MU-DL and MU-VL (dashed lines) are clearly visible on the AOTu anterior surface after fura-2 dextran staining of inter-tubercle neurons (overview). Dendrites of inter-tubercle neurons in the AOTu show calcium signals in response to light stimulations but not to dark controls. Stimulations of all three parts of the visual field clearly activate the AOTu. D, Time course of fura-2 calcium signals (percentage change in fluorescence ratio, average of three presentations for each bee, n = 10 bees) measured in the MU of the AOTu for stimulation of dorsal, lateral, or ventral parts of the visual field and for the dark control. E, Mean amplitude of activation (calculated at 4 s) for stimulations of dorsal, lateral, or ventral parts of the visual field and dark control in 10 bees. Different letters indicate significant differences in Wilcoxon's matched-pairs tests. do-ve, Dorsoventral; an-po, anteroposterior; do, dorsal; la, lateral.
- Figure 9.
Spatial coding in the honeybee AOTu. A, Example of activated pixels for three repetitions of dorsal, lateral, or ventral stimulations in the MU of the AOTu (overview). Significantly activated pixels are overlaid on a fluorescence photograph of the AOTu according to their amplitude of activation (ΔR) in false color. Density maps indicate the reliability of activated pixels: colors from dark red to white indicate pixels activated by one, two, or the three presentations of each stimulus. Overlay maps represent reliable pixels activated by the three stimulations of one part of the visual field in one color and the other part in another color (dorsal in magenta, lateral in blue, and ventral in green). Pixels activated by stimulations of both parts of the visual field are represented in white. These maps show good reproducibility of activated pixels for the same part of the visual field (density) and a segregation of activated pixels for different parts of the visual field (overlay, especially dorsal vs ventral). B, Comparison of the mean overlap between any two presentations of the same or of different stimuli in 10 bees. Activation patterns in the AOTu show clear spatial specificity, because presentations of the same stimulus present a higher overlap than presentations of different stimuli (Wilcoxon's signed-rank test). C, Four examples of the striking segregation between dorsal and ventral stimulations. White arrows in bee 1 and bee 4 indicate a dorsoventral segregation at the level of the axonal tract of vITT neurons (see also bee shown in A). D, The overlap index between dorsal and ventral stimuli (do-ve) is significantly lower than that between lateral and dorsal stimuli (do-la) or between lateral and ventral stimuli (la-ve) as shown by Wilcoxon's signed-rank test. E, Mean number of activated pixels in the MU-DL or MU-VL for stimulations of the dorsal, lateral, and ventral eye in 10 bees. In the MU-DL, ventral and lateral stimulations activated a significantly higher number of pixels than dorsal stimulation, whereas in the MU-VL, dorsal and lateral stimulations activated a higher number of pixels than ventral stimulation (Wilcoxon's signed-rank test).
- Figure 10.
Schematic representation of dorsoventral visual information flow both at the input (anatomical data, see Fig. 3) and at the output levels (physiological data, see Fig. 9) of the AOTu circuitry. Input, From medulla (Me) and lobula (Lo) to the AOTu via the AOT. The input pattern is enlarged in the boxed inset. Ventral eye: Ventral medulla neurons (ventral Me, dark green) project to the MU-VL (chiefly to its ventral part) and to the dorsal part of the MU-DL; ventral lobula neurons (ventral Lo, light green) project to the dorsal parts of the MU-DL and MU-VL. Dorsal eye: Dorsal medulla neurons (dorsal Me, red) only project to the ventral part of the MU-DL and to the VLU; dorsal lobula neurons (dorsal Lo, orange) project to the ventral part of the MU-DL and to the whole MU-VL. Output: From the ipsilateral AOTu to the contralateral AOTu via ITT. Ventral eye: Output inter-tubercle neurons in MU-DL are mostly activated by ventral eye stimulation. Dorsal eye: Output inter-tubercle neurons in MU-VL are mostly activated by dorsal eye stimulation. Thus, functionally, inter-tubercle output neurons in MU-DL seem to be mainly connected to the input neurons from the ventral eye (dark and light green arrows) to this compartment, whereas output neurons in MU-VL seem to be mainly connected to the input neurons from the dorsal eye to this compartment (orange arrows).
