Synganglion of Uloborus diversus.

(A.) 3D rendering of U. diversus (female) synganglion from averaged α-synapsin volume, composed of 909 z-planes, oblique posterior-lateral (left) and oblique anterio-lateral (right) views (B.) 3D rendering of α-synapsin (green) and DAPI stained (blue) synganglion, posterior, lateral and anterior views. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Leg neuropils.

(A.) 3D rendering of leg neuropils as annotated from averaged synapsin immunovolume with posterior oblique, anterior oblique, and dorsal views, left to right. (B.) Maximum intensity projections of z-planes 160-190 for leg neuropil expression of Synapsin (α-synapsin, gray), (i.) Serotonergic (α-5-HT, green), (ii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iii.) Cholinergic (α-ChAT, cyan), (iv.) Proctolin (α-Proctolin, yellow), (v.) FMRFamide (α-FMRFamide, red), (vi.) Cardioactive peptide (α-CCAP, cyan), and (vii.) AllatostatinA (α-AstA, green) immunoreactivity in the standard brain. Dotted perimeter marks the boundary of Leg Neuropil 1. (C.) Maximum intensity projections of z-planes 230-260. Dotted perimeter marks the boundary of Leg Neuropil 1. (D.) Maximum intensity projection showing Dopaminergic (α-TH, green) immunoreactivity, dotted perimeter marks the boundary of Leg Neuropil 1. Dopamingeric immunoreactivity is shown in a separate panel because this volume, unlike the other stains, is not aligned to the standard atlas. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Opisthosomal neuropil.

(A.) 3D rendering of the opisthosomal neuropil as annotated from averaged synapsin immunovolume with posterior oblique, anterior oblique, and dorsal views, left to right. (B.) Maximum intensity projections of z-planes 415-425 for opisthosomal neuropil expression of Synapsin (α-synapsin, gray), (i.) Serotonergic (α-5-HT, green), (ii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iii.) Cholinergic (α-ChAT, cyan), (iv.) Proctolin (α-Proctolin, yellow), (v.) FMRFamide (α-FMRFamide, red), (vi.) Cardioactive peptide (α-CCAP, cyan), and (vii.) AllatostatinA (α-AstA, green) immunoreactivity in the standard brain. Arrow in α-TDC2 subpanel marks tracks along perimeter of the opisthosomal neuropil, supplied anteriorly (brace). Arrowheads in α-TDC2 and α-Proctolin subpanels mark a neurite running centrally (C.) Maximum intensity projections of z-planes 448-465. Longitudinal and lateral projections form a ladder-like structure in the α-TDC2 subpanel (brace). A midline-crossing structure (arrow) is seen in the α-Proctolin subpanel. (D.) Maximum intensity projection showing Dopaminergic (α-TH, green) immunoreactivity in the opisthosomal neuropil. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Pedipalp, Blumenthal and Cheliceral neuropil.

(A.) 3D rendering of the pedipalp neuropil as annotated from averaged synapsin immunovolume with posterior oblique and anterior oblique views, left to right, and dorsal view, lower. (B.) Optical slices (z275) from the standard brain, containing a cross-section of the pedipalp neuropil (dashed line boundary), with expression of (i.) Synapsin (α-synapsin, gray), (ii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iii.) Cholinergic (α-ChAT, cyan), and (iv.) Proctolin (α-Proctolin, yellow) immunoreactivity. Arrow in α-Proctolin shows adjacent Proctolin+ somata. (C.) Optical slices (z360) from the standard brain, with a cropped and enlarged selection showing the Blumenthal neuropil (brace), with expression of (i.) Synapsin (α-synapsin, gray) and Proctolin (α-Proctolin, yellow) immunoreactivity above, and only (ii.) Proctolin below. A circular region of the Blumenthal neuropil (dashed oval) shows Proctolin immunoreactivity. (D.) 3D rendering of the cheliceral neuropil as annotated from averaged synapsin immunovolume with posterior oblique and anterior oblique views, left to right, and dorsal view, lower. (E.) Optical slices (z400) from the standard brain, containing a cross-section of the cheliceral neuropil (dashed line boundary), with expression of (i.) Synapsin (α-synapsin, gray), (ii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iii.) AllatostatinA (α-AstA, green), (iv.) Proctolin (α-Proctolin, yellow), (v.) Serotonergic (α-5-HT, green), and (vi.) Cholinergic (α-ChAT, cyan) immunoreactivity. Arrows in α-TDC2, α-AstA, and α-5-HT subpanels mark an unidentified medially adjacent region to the cheliceral neuropil which shows pronounced immunoreactivity for these antisera. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Ventral supraesophageal features.

Posterior and anterior stalk region expression of Synapsin (α-synapsin, gray), (i.) Serotonergic (α-5-HT, green), (ii.) Cholinergic (α-ChAT, cyan), (iii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iv.) Proctolin (α-Proctolin, yellow), (v.) AllatostatinA (α-AstA, green), and (vi.) Cardioactive peptide (α-CCAP, cyan) immunoreactivity in the standard brain, for: (A.) z-plane 461. Arrow in α-TDC2 subpanel marks tracks along perimeter of the opisthosomal neuropil, supplied anteriorly (brace). Top and bottom arrows in α-Proctolin subpanel represent posterior and anterior bridging immunoreactivity, respectively, in the stomodeal bridge (StB) area. Arrowhead in α-AstA subpanel marks a faint band of immunoreactivity across the midline. (B.) z-plane 490. Further ventrally, a bridging structure is also visible on the posterior end of the ventral supraesophageal, as seen in the α-Proctolin subpanel (brace). Cholinergic immunoreactivity is present in the protocerebral tract (PCT), arrowhead in α-ChAT subpanel. Bands of α-TDC2 immunoreactivity (arrow) which do not correspond to clear structures in the synapsin channel. (C.) z-plane 511. Arrowhead in α-ChAT subpanel marks prominent cholinergic immunoreactivity in the protocerebral tract (PCT), more clearly visible at this plane. In the α-AstA subpanel, arrow showing oxbow-like structure and pronounced innervation at the posterior midline, and central anterior stalk AstA+ innervation (brace). Arrows in α-TDC2 subpanel mark centrally located concentrations of TDC2 immunoreactivity anterior and lateral to the PCT. StB = stomodeal bridge, PCT = protocerebral tract. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Hagstone neuropil and mid-supraesophageal features.

(A.) 3D rendering of the hagstone neuropil as annotated from averaged synapsin immunovolume with posterior oblique, anterior oblique, and dorsal views, top to bottom. (B.-D.) Hagstone neuropil and posterior feature expression of Synapsin (α-synapsin, gray), (i.) Serotonergic (α-5-HT, green), (ii.) Proctolin (α-Proctolin, yellow), (iii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), and (iv.) AllatostatinA (α-AstA, green) immunoreactivity in the standard brain, for: (B.) z-plane 540. A circular tract pattern distinctive to the α-5-HT stain is visible in the posterior mid-supraesophageal (brace, α-5-HT subpanel). (C.) z-planes 565, with the boundary of hagstone neuropil (dashed perimeter). An umbrella-like innervation pattern is present posteriorly (brace), in both the α-5-HT and α-TDC2 subpanels. (D.) z-plane 585, with the boundary of hagstone neuropil (dashed perimeter). In the α-Proctolin subpanel, a brace marks a crescent-shaped zone of Proctolin immunoreactivity, which has also been observed in another spider species. PCT = protocerebral tract, MB = mushroom body. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Mushroom bodies.

(A.) 3D rendering of mushroom body neuropil as annotated from averaged α-synapsin volume, dorsal (top) and oblique posterior (bottom) (B.) Maximum intensity projection of averaged α-synapsin volume, showing the mushroom bodies to be the most strongly immunoreactive structure in the supraesophageal ganglion (C.) Optical sections of the supraesophageal ganglion from an averaged α-synapsin volume (ventral (top) to dorsal (bottom). The haft, body and head regions of the MB are labelled (D.) α-βTubulin3 (magenta) immunoreactivity aligned with α-Synapsin volume (gray) (compare to bottom portion of previous subfigure) showing the arching form of the mid-line spanning mushroom body bridge (E.) AllatostatinA immunoreactivity (α-AstA, green) present in the MB haft (pink dotted line marking location of α-synapsin immunoreactivity) with arrows pointing to innervation from the posterior side (F.) α-βTubulin3 (magenta) and α-Synapsin (green) immunoreactivity in the supraesophageal ganglion at the plane where the mushroom body hafts appear (round, intensely immunoreactive). Arrows mark a fiber tract flanking the haft which could be the origin of the innervation in the preceding subfigure (G.) Tripart tract entering at the mushroom body head to fuse with the tract descending through the MB. (H.). Cropped optical section from an individual Cholinergic stain (α-ChAT, cyan), of a plane just dorsal to the mushroom body heads, showing putative globuli cells (arrows) within the protrusion of the secondary visual pathway, DAPI stain (red). (I.) Cropped optical slice from standard brain (z609) at the level of mushroom body heads, with Synapsin (α-synapsin, gray), GABAergic (α -GAD, red), and merged immunoreactivity, with arrow indicating potential innervation of the globuli cells. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Visual pathways.

(A.) Immunostaining for α-HRP (magenta) for neuropil and use of DAPI (blue) for nuclei, arrows show the primary (1°) and secondary (2°) visual pathway extensions from the bulk of the supraesophageal tissue (B.) 3D renderings of synpasin-immunoreactivity in the dorsal supraesophageal ganglion, with tissue of the primary (1°) and secondary (2°) visual pathway visible. Brace shows path of secondary pathway input to mushroom body head. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Tonsillar neuropil.

(A.) 3D rendering of tonsillar neuropil as annotated from averaged synapsin immunovolume with posterior oblique, anterior oblique, and dorsal views, left to right (B.) Oblique horizontal optical section of supraesophageal ganglion with α-Synapsin (green) and α-βTubulin3 (magenta) immunoreactivity. The tonsillar neuropil is seen centrally, with the arrow denoting a fiber tract which passes medially across it. (C.) Ventral (z682) and dorsal (z700) views of the tonsillar neuropil, demarcated by dashed lines, from the standard brain, showing expression of Synapsin (gray), (i., vi.) Serotonergic (α-5-HT, green), (ii., vii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iii., viii.) Proctolin (α-Proctolin, yellow), (iv., ix.) AllatostatinA (α-AstA, green) and (v., x.) FMRFamide (α-FMRFamide, red) immunoreactivity. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Protocerebral bridge neuropil.

(A.) 3D rendering of protocerebral bridge (PCB) neuropil as annotated from averaged synapsin immunovolume, with posterior oblique, anterior oblique, and dorsal views, left to right. (B.) Ventral (z722) and dorsal (z730) views of the PCB, as demarcated by dashed lines, from the standard brain, showing expression of Synapsin (gray), (i., vi.) GABAergic (α-GAD, red), (ii., vii.) Octopaminergic/Tyraminergic (α-TDC2, magenta), (iii., viii.) Proctolin (α-Proctolin, yellow), (iv., ix.) Cholinergic (α-ChAT, cyan) and (v., x.) AllatostatinA (α-AstA, green) immunoreactivity. Arrows point to dorsal protocerebral commissure (dPCC). Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Arcuate body.

(A.) 3D rendering of arcuate body neuropil as annotated from averaged α-synapsin volume, posterior oblique, posterior, and anterior oblique views, left to right, respectively (B.) Individual 3D rendering of the ventral arcuate body lobe (ABv, dark green) and dorsal arcuate body lobe (ABd, light green), with magenta envelope representing space which would be occupied by the missing lobe in each image. Top row images are dorsal views, bottom row are oblique posterior (C.) Optical horizontal slices of α-synapsin immunoreactivity from the dorsal supraesophageal ganglion. Top image is ventral relative to the bottom, and shows the ventral arcuate body lobe (ABv), while the bottom image features both the ventral and dorsal arcuate body lobe (ABd). Each lobe contains an anterior (ant.) and posterior (pos.) section, marked with yellow dashed lines (D.) Ventral (top) and dorsal (bottom) views showing aligned image volumes of Proctolin (α-Proctolin, yellow), Crustacean Cardioactive Peptide (α-CCAP, cyan) and FMRFamide (α-FMRFamide, red) immunoreactivity, demonstrating distinct structures as well as overlapping innervation of the arcuate body layers (E.) α-βTubulin3 (magenta) and α-Synapsin (green) immunoreactivity in the arcuate body (ventral to dorsal as top to bottom, respectively), with arrows marking where pronounced fiber tracts pass through the arcuate body layers (F.) Dorsal view of arcuate body showing layering in ABv and ABd (brace), for Proctolin (α-Proctolin, yellow) and Octopaminergic/Tyraminergic (α-TDC2, magenta) immunoreactivity which have overlapping but distinct innervation patterns in the anterior ABd. (G.) Arrowheads marking patterning of individual units of α-AstA immunoreactivity in the arcuate body, suggestive of a columnar structure. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Arcuate body layers revealed by staining for specific neurosignaling populations.

Ventral (left column, odd roman numerals) and dorsal (right column, even roman numerals) horizontal optical section views of the arcuate body lobes: ventral AB, represented in the 3D rendering (dark green), and dorsal AB (light green). The perimeter of the whole arcuate body (ABv and ABd) is outlined (dashed line, magenta), as well as the boundary (dashed line, green) between ABv (labelled internally as “v.”) and ABd (labelled internally as “d.”). Immunoreactivity for the following targets is shown: GABAergic (α-GAD) (i., ii.), arrows in ventral image marking neurites traversing arcuate body layers from the posterior cell layer, arrow and arrowhead in dorsal image mark posterior ABd innervation patterns, brace in dorsal image marks a thicker innervation pattern in the anterior ABd sublayer. Cholinergic (α-ChAT) (iii., iv.), in the ventral image the brace marks a fine puncta layer in the anterior ABv sublayer, in the dorsal image an arrowhead marks pronounced varicosities in the dorsal aspect of the anterior ABv, while a brace marks the flagstone-pattern innervation seen in the anterior sublayer of the ABd. Dopaminergic (α -TH) (v., vi.), in the dorsal image an arrow marks a layer of innervation in the posterior ABd. Serotonergic (α -5-HT) (vii., viii.), in the ventral image an arrow marks a faint layer of immunoreactivity in the posterior ABv and a brace marks broad, fine innervation in the anterior ABv. In the dorsal image a brace marks the width of the anterior ABd layer showing flagstone-pattern innervation. Octopaminergic/Tyraminergic (α-TDC2) (ix., x.), in the ventral image an arrow marks dense varicosities in the anterior ABd sublayer. In the ventral image a brace marks pronounced flagstone-pattern innervation in the anterior ABv sublayer. AllatostatinA (α-AstA) (xi., xii.), in the ventral image a brace marks the innervation of the anterior ABv. Proctolin (α-Proctolin) (xiii., xiv.), in the ventral image an arrowhead marks intense immunoreactive terminals at the posterior ABv, and the stacked braces define the boundaries of innervation to the posterior (top) and anterior (bottom) sublayers of the anterior ABv. In the dorsal image an arrowhead marks an example of garland-like varicosities visible at the dorsal end of the ABv. Crustacean Cardioactive Peptide (α-CCAP) (xv., xvi.), in the ventral image stacked braces mark the posterior (top brace) and anterior (bottom brace) layers of the ABv. FMRFamide (α-FMRFamide) (xvii., xviii.), in the ventral image stacked braces mark the boundaries of posterior (top) and anterior (bottom) layers of the ABv. In the dorsal image the brace marks innervation to the posterior ABd layer. Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)

Summary of U. diversus neuropils.

(A.) Posterior oblique, dorsal and anterior oblique views of 3D renderings of the standard U. diversus brain with annotations of major established and novel neuropils throughout the subesophageal and supraesophageal masses (B.) 3D renderings of the standard U. diversus brain with annotations of potential central complex constituents in shades of green (protocerebral bridge, arcuate body ventral and dorsal lobes, tonsillar neuropil), also showing the mushroom body (purple) (C.) 3D neuropil renderings from of neuropils of the central complex as found in the insects Rhyparabia maderae, Scarabaeus lamarcki, and Manduca sexta (images from insectbraindb.org). Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right. (See eLife edition for higher-resolution image.)