Journal: bioRxiv

Article Title: Distributed neural computation and the evolution of the first brains

doi: 10.1101/2025.10.03.680388

Figure Lengend Snippet: a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a Par3 antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).

Article Snippet: Primary antibodies used: Par3 (St. John’s Laboratory #STJ94951, 1:200), pERK (Cell Signaling Technologies #4370T, 1:200) , FMRFamide (EMDMillipore #AB15348, 1:1000) .

Techniques: Staining, Electron Microscopy