Synganglion of Uloborus diversus.

(A.) 3D rendering of U. diversus (female) synganglion from averaged α-synapsin volume, 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 (C.) Sequence of horizontal optical slices from averaged α-synapsin (gray) volume with averaged DAPI stains (blue), from ventral subesophageal ganglion (left) to dorsal end of supraesophageal ganglion (right). Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = lateral, M = medial.

Overview of averaged α-synapsin immunoreactivity in whole-mount synganglion.

Sequence of optical horizontal sections from averaged α-synapsin volume, with top-right insets showing position of respective slice in a 3D full volume rendering (A. – I.) Subesophageal ganglion, beginning ventrally (A.) and progressing dorsally until (I.). Notable features include the leg neuromeres (LN1-4, for respective legs 1-4), pedipalpal neuropil (PdN), cheliceral neuropil (ChN), opisthosomal neuropil (OpN, which is still visible until (L.)), and the esophageal passage. (J. – T.) Supraesophageal ganglion, with marked features including the stomodeal bridge (STb), protocerebral tract, protocerebral commissure (PCC), hagstone neuropil (HsN), mushroom body (haft, body, and head), tonsillar neuropil, arcuate body (ventral and dorsal lobes, ABv and ABd, respectively), and protocereral bridge (PCB).

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.

Visual pathways.

(A.) Immunostaining for α-HRP (magenta) for neuropil and use of DAPI (blue) for nuclei, arrows show the primary (⍰) and secondary (⍰⍰) 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 (⍰) and secondary (⍰⍰) visual pathway visible.

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 relatively ventral 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.

Arcuate body layers revealed by staining for specific neurosignaling populations.

Ventral (left column) and dorsal (right column) horizontal optical section views of the arcuate body (perimeter of whole arcuate body marked by dashed line) for GABAergic (α-GAD), Cholinergic (α-ChAT), Dopaminergic (α-TH), Serotonergic (α-5-HT), Octopaminergic/Tyraminergic (α-TDC2), AllatostatinA (α-AstA), Proctolin (α-Proctolin), Crustacean Cardioactive Peptide (α-CCAP), and FMRFamide (α-FMRFamide) immunoreactivity.

Centrally-located, 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 and dorsal views of the tonsillar neuropil, as demarcated by dotted lines. Synapsin (gray), Serotonergic (α-5-HT, green), Octopaminergic/Tyraminergic (α-TDC2, magenta), Proctolin (α-Proctolin, yellow), AllatostatinA (α-AstA, green) and FMRFamide (α-FMRFamide, red) immunoreactivity.

Protocerebral bridge neuropil.

(A.) 3D rendering of protocerebral bridge neuropil as annotated from averaged synapsin immunovolume. (B.) Ventral and dorsal views of the PCB, as demarcated by dotted lines. Synapsin (gray), GABAergic (α-GAD, red), Octopaminergic/Tyraminergic (α-TDC2, magenta), Proctolin (α-Proctolin, yellow), Cholinergic (α-ChAT, cyan) and AllatostatinA (α-AstA, green) immunoreactivity. Arrows point to posterior protocerebral commissure.

A potential central complex in U. diversus

(A.) 3D renderings of averaged U. diversus synganglion with annotations of potential central complex constituents in shades of green (protocerebral bridge, arcuate body lobes, tonsillar neuropil), also showing the mushroom body (purple) (B.) 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)