1. Polyploidy and the Cellular and Areal Diversity of Rat Cortical Layer 5 Pyramidal Neurons 
Johanna Sigl-Glöckner, Michael Brecht 
Cell Reports 
Volume 20, Issue 11, Pages (September 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 2575-2583DOI: (10.1016/j.celrep.2017.08.069)
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3. Figure 1
Neurons in L5 of Visual Cortex Have Larger Nuclei and Somata than Neurons in Other Layers
(A) Coronal section of primary visual cortex (V1) stained with DAPI (left) and an anti-NeuN antibody (middle) revealing neuronal somata (overlay, right). White squares indicate the position of sections shown in (B) (L4) and in (C) (L5).
(B) Section through cortical L4 of V1 stained with DAPI and NeuN.
(C) Section through cortical L5 of V1.
(D) Soma size versus nucleus size for L2 (purple, n = 86), L3 (green, n = 92), L4 (blue, n = 124), L5 (red, n = 348), and L6 neurons (orange, n = 107). Dashed lines indicate a linear fit.
(E) Frequency distribution histograms for soma and nucleus size of cortical layers 2 to 6.
Scale bars represent 100 μm (A) and 30 μm (B and C).
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4. Figure 2
Integrated DAPI Fluorescence and Chromocenter Counts for Cortical Layers 2 to 6
(A) Schematic illustrating how integrated fluorescence is measured. Z-stack of a nucleus stained with DAPI. Fluorescence of each optical plane is integrated, normalized, and summed up across all sections.
(B) Frequency distribution and univariate plot of DAPI fluorescence of neurons in L2 (purple, n = 86), L3 (green, n = 92), L4 (blue, n = 126), L5 (red, n = 347), and L6 (orange, n = 107) and non-neuronal cells (gray, n = 556; NeuN−) in V1. Cellular fluorescence measurements are normalized to the mean normalized fluorescence of non-neuronal cells (by definition = 1) in each section analyzed.
(C) L5 neuron stained with NeuN (red, top left) and DAPI (blue, bottom left). White arrows indicate 10 chromocenters in the displayed section. In total, this nucleus has 27 chromocenters.
(D) Non-neuronal nucleus, as in (C). Note the absence of NeuN staining (red channel). There are 6 chromocenters in the displayed section. In total this nucleus has 19 chromocenters.
(E) Frequency distributions and univariate plots of chromocenter counts of cells in (B).
Dashed lines indicate mean integrated fluorescence and chromocenter counts of non-neuronal cells (1.0 by definition and 20) and twice this number. Black crosses indicate mean ± SD. Black lines indicate significant pairwise comparisons (Kruskal-Wallis ANOVA, p < 0.05). Scale bars represent 5 μm. See also Figure S1.
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5. Figure 3
Integrated DAPI Fluorescence and Chromocenter Counts of Identified Cell Types in L5
(A) Confocal image of rat V1 L5 stained with DAPI (blue), NeuN (red), an antibody against the transcription factor Ctip2 (green), and overlay.
(B) Same as (A), stained with DAPI (blue), NeuN (red), an antibody against parvalbumin (yellow), and overlay.
(C) Distribution and univariate plot of integrated DAPI fluorescence of neurons negative for the transcription factor Ctip2 (Ctip2−, blue, n = 207), neurons positive for the transcription factor Ctip2 (Ctip2+, green, n = 142), parvalbumin-positive interneurons (PV+, yellow, n = 54), and non-neuronal cells (NN, gray, n = 365).
(D) Chromocenter counts of identified cells in (C).
Dashed lines indicate mean integrated fluorescence and chromocenter counts (by definition 1.00 and 20) and twice this number. Black crosses indicate mean ± SD. Scale bar represents 20 μm. See also Figure S2.
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6. Figure 4
Integrated DAPI Fluorescence and Chromocenter Counts of L5 Cells in Three Different Cortical Areas
(A) Frequency distribution and univariate plot of integrated DAPI fluorescence of Ctip2− (blue), Ctip2+ (green), and non-neuronal cells (NN, gray) in L5 of three cortical areas with large, medium, and small mean neuronal size: primary AI (n = 261 Ctip2−, n = 156 Ctip2+, n = 101 NN), V1 (n = 207 Ctip2−, n = 140 Ctip2+, n = 98 NN), and granular retrosplenial cortex (RSG; n = 42 Ctip2−, n = 49 Ctip2+, n = 94 NN).
(B) Chromocenter counts of cells in (A).
Dashed lines indicate mean integrated fluorescence and chromocenter counts (by definition 1.00 and 20) and twice this number. Black crosses indicate mean ± SD. See also Figures S3 and S4.
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7. Figure 5
Fluorescent In Situ Hybridization of Telomeres in Suspended Interphase Nuclei of Cortical L4 and L5
(A) Suspended nuclei of L4 (top) and L5 (bottom) stained with DAPI (blue, left), a PNA probe against rat telomere (TelC; green, middle), and overlay (right). Insets show a representative L4 nucleus (top, 11 μm diameter, 53 telomere spots) and a large L5 nucleus (bottom, 30 μm diameter, 104 telomere spots). White arrows indicate green telomere spots.
(B) Frequency distribution and univariate plot of nucleus size for L4 (blue, n = 84) and L5 (red, n = 126).
(C) Telomere counts for cells in (B).
Dashed lines indicate the mean number of telomeres of L4 (52) and twice this number. Black crosses indicate mean ± SD. Stars indicate significant pairwise comparisons (Kruskal-Wallis ANOVA, p < ). Scale bars represent 25 and 5 μm (all insets). See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
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