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

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Characterizing in vivo para-uterine method for imaging cortical neurons, related to Figure 3 (A) Mean (red dot) of embryonic weights on each day (black dots) ranges from 0.17 g at E13.5 to 1.3 g at E18.5. N = number of embryos. (B) Stability of in vivo para-uterine two-photon imaging of cortical neurons in embryos is similar to stability of two-photon imaging of cortical neurons in adult mice. Movement per frame (recorded from 5 to 10 Hz), averaged per recording, computed via rigid motion correction between frames. In vivo embryonic recordings were made as schematized in Figure 3 A. Adult recordings were made in head-fixed Rbp4-Cre mice, injected with AAV expressing Cre-dependent GCaMP. Probability: Wilcoxon rank-sum test; n = number of recordings from 3 (E13.5), 9 (E14.5), 5 (E15.5), 4 (E16.5), 5 (E17.5), and 6 (E18.5) embryos and 3 adult mice. (C–E) Embryonic blood flow does not change following 5 h of imaging, but degrades rapidly following severing of the umbilical cord. Blood flow at the surface of the brain was imaged in visible light prior to and following 5 h of imaging (C). The difference in blood flow is quantified (left), at E14.5 (D) and E18.5 (E). Blood flow at the surface of the brain was also imaged immediately prior to, and 10 min following the severing of the umbilical cord. The difference in blood flow is quantified (right), at E14.5 (D) and E18.5 (E). Box-and-whiskers: distribution of changes in blood flow across each time window, as box (25–75 percentile) and whisker (5–95 percentile); red lines: median. Probability: Wilcoxon rank-sum test. (F) Temperature of embryo stabilized para-uterine under the two-photon microscope . Infrared image is aligned with a visible light image, where the embryo can be observed within the holder (as schematized in Figure 3 A). The 36.5°C marker labels the embryo, , which is visible through the opening in the holder allowing for the exit of the umbilical cord. The second 50°C marker labels the objective heater, providing a secondary source of heat during imaging. Image was taken following 5 h of imaging. (G) Fluorescence of Rbp4-Cre neurons is significantly increased over background fluorescence from E13.5 to E18.5. Mean cellular fluorescence, normalized by the pixel size of each cell, compared against the mean pixel fluorescence within three background regions selected within the imaging window, collected across all neurons, across all embryonic days (left) and on each embryonic day (right). Black circles: ratio of fluorescence within individual background regions in each imaging plane compared to each other; gray circles: ratio of fluorescence within individual neurons compared to each background region in the same imaging plane; box-and-whiskers: distributions across background fluorescence ratios (black) and cellular fluorescence ratios (gray) as box (25–75 percentile) and whisker (10–90 percentile); black and red line: median; blue line and text: mean. Probability: Wilcoxon rank-sum test comparing cellular fluorescence ratios to background fluorescence ratios. Recordings from 3 (E13.5), 9 (E14.5), 5 (E15.5), 4 (E16.5), 5 (E17.5), and 6 (E18.5) embryos. (H) Imaging region shown with respect to the embryonic brain at E13.5 (left), E15.5 (middle), and E18.5 (right). Imaging region was centered over the posterior dorsal pallium. Images taken from Allen Developing Brain Atlas ( http://atlas.brain-map.org/ ).

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 (UPLSAPO40×S, Olympus) objectives.

Techniques: In Vivo, Imaging, Injection, Expressing, Whisker Assay, Microscopy, Marker, Fluorescence

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Rbp4-Cre neurons form active circuits already at E14.5 (A) Left: schematic of NMDA+AMPA injection during in vivo embryonic two-photon imaging. Right: Rbp4-Cre neurons; color: normalized calcium activity). (B) Change in fluorescence before (Pre) and after (Post) application of either cortex buffer (blue) or NMDA+AMPA (red). Wilcoxon signed rank test. n = number of Rbp4-Cre neuron ROIs from 3 E14.5 and 3 E18.5 embryos ( Figure S7 ). (C) Pairwise correlations of Rbp4-Cre neurons’ calcium activity, that are significantly greater than random ( Figure S7 ). Shuffled data is on the left. Filled circles: correlations; gray shading: distribution; red line: median. Bars: percent of neuron pairs with correlations significantly greater than random (red). Wilcoxon rank-sum test. n = pairs of Rbp4-Cre neurons recorded from 3 (E13.5), 9 (E14.5), 5 (E15.5), 4 (E16.5), 5 (E17.5), and 6 (E18.5) embryos. (D) Immunostaining of Rbp4-Cre neurons (green), Map2 (dendrites, red), NF (axons, white), Hoechst (blue). (E) Left: schematic in vivo para-uterine imaging using 3D acousto-optic two-photon microscope. Top middle: mean projections around each soma. Bottom middle: Three zoomed examples (red outline, top middle). Right: Δf/f activity from examples. Cells 1 and 3 have high correlation. (F) Pairwise correlations of E14.5 Rbp4-Cre neurons’ activity that are significantly greater than random, within and across layers. Dots: pairwise correlations; box (25–75 percentile) and whisker (5–95 percentile); line: median. Scale bars: 10 μm (A), 30 μm (top, D), 100 μm (bottom, D), 20s and 5 %ΔF/F (E). See also Figure S7 .

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 (UPLSAPO40×S, Olympus) objectives.

Techniques: Injection, In Vivo, Imaging, Activity Assay, Fluorescence, Immunostaining, Microscopy, Whisker Assay

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Perturbing autism-associated genes selectively in Rbp4-Cre neurons disrupts circuit organization and activity during embryonic development (A) Expression (circles) of selected genes associated with autism spectrum disorder in the three Rbp4-Cre neuron types and adult L5-PN types. Radius of circles: fraction of cells expressing the gene; color of circles: mean normalized transcripts per cell (log 2 ). (B) Fraction of genes with a mean transcript count greater than the number of transcripts shown on the x axis, for all genes (black), and genes associated with autism spectrum disorder (magenta) in Rbp4-Cre neurons (top) and adult L5-PNs (bottom). Inset: Fold change of autism-associated gene expression compared to all genes in embryos and adult. (C) Immunostaining of cortex of Rbp4-tdTomato-Chd8 +/− (top) and Rbp4-tdTomato-Grin2b +/− (bottom) mice ( Figure S8 ). Rbp4-Cre neurons (red), Bcl11b, (white), Hoechst (blue). (D) Normalized depths of Rbp4-Cre neurons (as in Figure 2 A) in Rbp4-tdTomato (WT) and the two mutant (top, Chd8 +/− ; bottom, Grin2b +/− ) embryos ( Figure S9 ). 125 neurons from each mouse line, sampled at random. χ 2 test. (E) Mating strategy to generate Rbp4-GCaMP6s-tTA2-Chd8 +/− (Chd8 +/− ) and Rbp4-GCaMP6s-tTA2-Grin2b +/− (Grin2b +/− ) embryos. (F) Example recordings from Rbp4-Cre neurons’ dendrites in Rbp4-GCaMP6s-tTA2 (WT), Grin2b +/− , and Chd8 +/− embryos at E16.5 using 3D acousto-optic two-photon microscope. (G) Distribution of activity in E16.5 embryos, shown in log-scale, for WT and two mutant genotypes. Circles: activity of each neurite; red line: median; shading: distribution. Wilcoxon rank-sum test. n = number of neurites. (H) Immunostaining of local patches of cortical disorganization in Rbp4-tdTomato-Chd8 +/− and Rbp4-tdTomato-Grin2b +/− mice, at E18.5. Rbp4-Cre neurons (red), Bcl11b, (white), Hoechst (blue). (I) Fraction of mutant mice, of each genotype, showing at least one patch, summed across E16.5 to E18.5. Red line: Average across all four genotypes. Data from 14 (Rbp4-tdTomato-Chd8 +/− ), 11 (Rbp4-tdTomato-Chd8 −/− ), 9 (Rbp4-tdTomato-Grin2b +/− ), and 6 (Rbp4-tdTomato-Grin2b −/− ) embryos. Fisher’s exact test (p = 0.05, prior to Bonferroni correction). (J) Fraction of neurons within the superficial layer that are located within patches of disorganization, in embryos with at least one patch. n = number of superficial layer neurons on each embryonic day. Scale bars: 20 μm (C), 25s and 25 %ΔF/F (F), 50 μm (H). See also Figures S8 and .

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 (UPLSAPO40×S, Olympus) objectives.

Techniques: Activity Assay, Expressing, Gene Expression, Immunostaining, Mutagenesis, Microscopy