Journal: bioRxiv

Article Title: Embryonic cortical layer 5 pyramidal neurons form an active, transient circuit motif perturbed by autism-associated mutations

doi: 10.1101/2022.08.31.506080

Figure Lengend Snippet: ( A ) Left: schematic of injection of a mixture of cortex buffer with NMDA and AMPA (NMDA + AMPA) during in vivo embryonic two-photon para-uterine imaging. Right: Rbp4-Cre neurons before and after injection of either cortex buffer (control) or NMDA+AMPA at E14.5 and E18.5. ( B ) Change in fluorescence from baseline both before (Pre) and after (Post) application of either cortex buffer (blue) or NMDA + AMPA (red). Probability: Wilcoxon signed rank test; n = number of Rbp4-Cre ROIs from 3 (E14.5) and 3 (E18.5) embryos. ( C ) Correlations of spontaneous calcium activity, that are significantly greater than random, between pairs of Rbp4-Cre neurons on each embryonic day from E14.5 to E18.5. Filled circles: pairwise correlations; dark gray shading: distribution; red line: median. Random: pairwise correlations modelling the distribution of correlations from shuffling events in each Rbp4-Cre neurons; light gray shading: distribution. Bars: percent of neuron pairs with correlations greater than random (red) on each embryonic day. Probability: Wilcoxon rank-sum test. n = 425 (E14.5), 575 (E15.5), 187 (E16.5), 2517 (E17.5) and 2171 (E18.5) pairs of embryonic neurons recorded from 3 (E13.5), 9 (E14.5), 5 (E15.5), 4 (E16.5), 5 (E17.5), and 6 (E18.5) embryos. ( D ) Activity across somas and neurites, in the two active phases, within each spatial layer (blue: upper layer; gray: middle layer; beige: lower layer; as defined in ). ( E ) Immunohistochemical staining of Rbp4-Cre neurons (stained using GFP antibody, green), dendrites (stained using Map2 antibody, red), and axons (stained using NF antibody, white) at E14.5 and E18.5, within the developing cortex, counterstained with Hoechst (blue). ( F ) Left: schematic representation of in vivo para-uterine imaging of embryonic cortex using a 3D acousto-optic two photon microscope, allowing for random-access to cells within both layers, simultaneously, at E14.5. Top middle: mean projections of three-dimensional volumes around each neuronal soma in the three-dimensional imaging field are shown, with three examples (red outline) detailed (bottom middle). Examples are selected from both layers. Right: Δf/f activity traces from example neuronal somas (as labeled on left). Cells 1 and 3 are an example of a pair between layers with high correlation. ( G ) Correlations of spontaneous calcium activity, that are significantly greater than random, recorded as shown schematically in ( F ) between pairs of neurons within the same layer (left) and pairs of neurons in different layers (right). Dots: pairs of neurons; red box-and-whiskers: distribution as box (25-75 percentile) and whisker (5-95 percentile); red line: median. Scale bars: 10 μm (A), 30 μm (top, E), 100 μm (bottom, E), 20s and 5 %ΔF/F (F).

Article Snippet: A spinning disc microscope (Axio Imager M2 upright microscope, Yokogawa CSU W1 dual camera T2 spinning disk confocal scanning unit, Visitron VS-Homogenizer on an Olympus IXplore Spin confocal spinning disc microscope system) was used to image immunohistologically-stained slides, using 20X (UPLSAPO20X, Olympus) and 40X (UPLSAPO40XS, Olympus) objectives.

Techniques: Injection, In Vivo, Imaging, Control, Fluorescence, Activity Assay, Immunohistochemical staining, Staining, Microscopy, Labeling, Whisker Assay