Journal: Frontiers in Cellular Neuroscience

Article Title: Oligodendrocyte arrangement, identification and morphology in the developing superior olivary complex

doi: 10.3389/fncel.2025.1561312

Figure Lengend Snippet: Oligodendrocyte distribution in the developing SOC nuclei. (A) Top: Olig2 (magenta) and VGluT1 (green) co-labeling in transversal brainstem sections from P5, P14 and P54 gerbils. VGluT1 labeling indicates the medial nucleus of the trapezoid body (MNTB), medial superior olive (MSO) and lateral superior olive (LSO). The VGluT1-negative dorsal LSO area (dLSO) represents the wedge between the two VGluT1-positive LSO limbs. The control region is located in a VGluT1-negative area dorsal to the MNTB. Scale bars equal 200 μm. Bottom: Olig2 labeling from images in the top row. (B) Distribution of oligodendrocytes in the MNTB, MSO, LSO, dLSO and control area in P5, P14 and P54 gerbils. Average normalized density of counted oligodendrocytes determined from single sections per nucleus and age group, visualized by normalized gray values. Number ( n ) of sections/animals/litters (age group): 5/3/2 (P5), 14/2/2 (P14), 18/6/3 (P50-59, grouped to P54). (C) Normalized intensity as a function of relative location at P5 (gray), P14 (light blue) and P54 (dark blue) obtained from normalized density plots of single sections [ n for gerbil see (B) , n of sections/animals/litters for Etruscan shrew: 3/3/2]. Intensity profiles are shown in medio-lateral orientation for the MNTB and control region, dorso-ventral orientation for the MSO and dLSO and dorsomedial-dorsolateral orientation in the LSO. Lines show average values and shaded areas the SEM. Note that for the MNTB data from adult Etruscan shrews was added (red). Average intensities between 5 and 15% (medial, dorsal or dorsomedial) and 85–95% (lateral, ventral or dorsolateral) of the relative location were statistically compared. MNTB: gerbil P5 p = 0.923 U , P14 p = 0.929 U , P54 p = 0.003 U ; Etruscan shrew p = 0.415 M . MSO: P5 p = 0.258 U , P14 p = 0.689 U , P54 p = 0.331 M . LSO: P5 p = 0.055 M , P14 p < 0.001 U , P54 p = 0.051 M . dLSO: P5 p = 0.414 M , P14 p < 0.001 M , P54 p = 0.003 M . Control: P5 p = 0.938 U , P14 p = 0.923 M , P54 p = 0.704 M . U = unpaired t -test, M = Mann Whitney U test.

Article Snippet: Sections were then incubated for 3 days at 4°C in blocking solution containing the primary antibodies: microtubule-associated protein 2 (MAP2), chicken, 1:1000, Origene (Herford, Germany), Cat# TA336617; myelin basic protein (MBP), rat, 1:500, Abcam (Cambridge, UK), Cat# ab7349, RRID:AB_305869; Olig2, rabbit, 1:3000, ProteinTech (Planegg-Martinsried, Germany), Cat# 13999-1-AP, RRID:AB_2157541; SOX10, goat, 1:1000, R&D Systems (Wiesbaden, Germany), Cat# AF2864, RRID:AB_442208; S100, mouse, 1:5000, Thermofisher (Darmstadt, Germany), Cat# MA5-12969, RRID:AB_10983883; vesicular glutamate transporter 1 (VGluT1), guinea pig, 1:2000, Synaptic Systems (Goettingen, Germany), Cat# 135304, RRID:AB_887878.

Techniques: Labeling, Control, MANN-WHITNEY

Journal: Frontiers in Cellular Neuroscience

Article Title: Oligodendrocyte arrangement, identification and morphology in the developing superior olivary complex

doi: 10.3389/fncel.2025.1561312

Figure Lengend Snippet: S100 labeled oligodendrocytes in the developing SOC. (A) Example overviews of S100 labeling in transversal SOC sections from P5, P10 and P54 gerbil. Scale bars equal 150 μm. (B) Left: S100 (green) co-labeling with SOX10 (magenta) and Olig2 (cyan) in the gerbil MNTB at P54. Right: Maximum projection showing S100-positive cells from image on the left at higher magnification. Scale bars equal 20 μm (left) and 15 μm (right). (C) Cell density of S100-positive cells in the four given regions at P10, P14, and P54. Dots show values per section and rhombs median densities. Comparison between regions: P10 p < 0.001 K , P14 p = 0.678 A , P54 p = 0.552 A and age groups: P10 vs. P14: MNTB, control p > 0.999 K , MSO p = 0.689 K , LSO p = 0.017 K ; P10 vs. P54 and P14 vs. P54: p < 0.001 A . Number of sections/animals/litters per age group: 3/3/2. (D) Percentage of cells detected with S100, Olig2 and/or SOX10, SOX10 and S100, Olig2 and S100 or Olig2/SOX10/S100 triple labeling in the SOC and control region at P10, P14, and P54. Number of n as in (C) . (E) Example overviews of S100 (green), MBP (magenta), and VGluT1 (cyan) co-labeling in sagittal sections of the MNTB at P10 and P54. Squares mark regions of defined size (20 × 20 μm) where S100/MBP single and double labeled axons were counted. Insets show S100 and MBP labeling in the marked area at higher magnification. (F) Methodology to quantify the number of S100 (green) and Olig2 (magenta) single and double labeled cells in proximity of VGluT1-positive calyx-MNTB neuron synapses (gray). The sampling area where cells were considered in calyx proximity (outer white circle), was determined for each cell individually using the sum of the circle diameter surrounding a given calyx (inner white circle) and the average diameter of a S100-positive cell. Cells were counted and assigned as S100 (blue dot) or Olig2 single labeled (pink dot) or double labeled cells (blue and pink dot). Scale bar equals 10 μm. (G) Number of oligodendrocytes in calyx proximity at P10 and P54. Counts per calyx are shown by unfilled dots and represent the respective sum of all cell types. Filled dots show average values of all oligodendrocyte types (black), single S100- (dark gray) or Olig2-positive (light gray framed) and double labeled (magenta) cells. Number of sections/animals/litters/calyces (age group): 11/2/1/88 (P10), 11/2/1/89 (P54). (H) Average percentage of oligodendrocyte cell types in calyx proximity at P10 and P54. Number of n as in (G) .

Article Snippet: Sections were then incubated for 3 days at 4°C in blocking solution containing the primary antibodies: microtubule-associated protein 2 (MAP2), chicken, 1:1000, Origene (Herford, Germany), Cat# TA336617; myelin basic protein (MBP), rat, 1:500, Abcam (Cambridge, UK), Cat# ab7349, RRID:AB_305869; Olig2, rabbit, 1:3000, ProteinTech (Planegg-Martinsried, Germany), Cat# 13999-1-AP, RRID:AB_2157541; SOX10, goat, 1:1000, R&D Systems (Wiesbaden, Germany), Cat# AF2864, RRID:AB_442208; S100, mouse, 1:5000, Thermofisher (Darmstadt, Germany), Cat# MA5-12969, RRID:AB_10983883; vesicular glutamate transporter 1 (VGluT1), guinea pig, 1:2000, Synaptic Systems (Goettingen, Germany), Cat# 135304, RRID:AB_887878.

Techniques: Labeling, Comparison, Control, Sampling

Journal: Frontiers in Cellular Neuroscience

Article Title: Oligodendrocyte arrangement, identification and morphology in the developing superior olivary complex

doi: 10.3389/fncel.2025.1561312

Figure Lengend Snippet: Olig2- and SOX10-based oligodendrocyte identification in the developing SOC. (A) Example co-labeling of VGluT1 (blue), Olig2 (magenta), and SOX10 (green) in a transversal brainstem section from a P5 gerbil. Squares show areas of cell counting comprising a 72 × 72 μm region in the MNTB, MSO, medial and lateral LSO as well as in the control region. Note that data from the LSO was pooled. Scale bar equals 200 μm. (B) Insets from the marked areas in (A) show Olig2 and SOX10 co-labeling in the MNTB, MSO, medial (med.) and lateral (lat.) LSO and control region. Additionally, co-labeling in the MNTB of mature Etruscan shrew (MNTB_E) is depicted. (C) Percentage of oligodendrocyte cells detected with Olig2 (magenta), SOX10 (green) or both (gray) markers at P5, P14, and P36 in the MNTB, MSO, LSO and control area in gerbil. For the MNTB, percentages from adult Etruscan shrew (E) are added. Total cell number is given above for each age group. Comparison of single SOX10-positive fraction between age groups: LSO p = 0.21 K , MSO and MNTB p < 0.001 K , control: p = 0.005 K . Comparison of single Olig2-positive fraction before vs. after hearing onset: P5 vs. P14: MNTB A , MSO K , LSO K p < 0.001, control K p = 0.1. Number of sections/animals/litters (age group): 8/3/2 (P5), 16/2/1 (P14), 16/2/1 (P36), 3/3/2 (Etruscan shrew). (D) Normalized density of oligodendrocytes either detected by Olig2 (magenta) or Olig2/SOX10 co-labeling (gray) in gerbil. For the MNTB, data from adult Etruscan shrew (E) is added. Comparison of single Olig2 vs. Olig2/SOX10 double-labeled cells: P5: MNTB p > 0.99 A , MSO p = 0.347 A , LSO p < 0.001 K , control p = 0.187 A ; P14: MNTB p = 0.143 A , MSO p < 0.001 A , LSO p = 0.166 K , control p = 0.976 A . Number of n as in (C) .

Article Snippet: Sections were then incubated for 3 days at 4°C in blocking solution containing the primary antibodies: microtubule-associated protein 2 (MAP2), chicken, 1:1000, Origene (Herford, Germany), Cat# TA336617; myelin basic protein (MBP), rat, 1:500, Abcam (Cambridge, UK), Cat# ab7349, RRID:AB_305869; Olig2, rabbit, 1:3000, ProteinTech (Planegg-Martinsried, Germany), Cat# 13999-1-AP, RRID:AB_2157541; SOX10, goat, 1:1000, R&D Systems (Wiesbaden, Germany), Cat# AF2864, RRID:AB_442208; S100, mouse, 1:5000, Thermofisher (Darmstadt, Germany), Cat# MA5-12969, RRID:AB_10983883; vesicular glutamate transporter 1 (VGluT1), guinea pig, 1:2000, Synaptic Systems (Goettingen, Germany), Cat# 135304, RRID:AB_887878.

Techniques: Labeling, Cell Counting, Control, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Oligodendrocyte arrangement, identification and morphology in the developing superior olivary complex

doi: 10.3389/fncel.2025.1561312

Figure Lengend Snippet: Oligodendrocyte arrangement in the developing MSO. (A) MAP2 (blue), VGluT1 (green) and Olig2 (magenta) co-labeling in the MSO from P5, P10 and P34 gerbil. Rectangles show the respective profile area of 359 × 72 μm. Scale bar equals 50 μm. (B) Overlaid fluorescence intensity profiles of arbitrary units (a.u.) of MAP2 (blue), VGluT1 (green) and Olig2 (magenta) at P5, P10 and P36. Medial to lateral from left to right. (C) Intensity profiles of Olig2 at the given postnatal ages. Blue bar represents the location of MSO somata based on MAP2 labeling. Number of sections/animals/litters (age group): 5/3/2 (P5), 23/4/2 (P7), 13/3/2 (P10), 16/4/2 (P12), 26/2/2 (P14), 26/3/2 (P17), 18/4/3 (P21), 22/4/4 (P34-38; grouped to P36), 18/6/3 (P54).

Article Snippet: Sections were then incubated for 3 days at 4°C in blocking solution containing the primary antibodies: microtubule-associated protein 2 (MAP2), chicken, 1:1000, Origene (Herford, Germany), Cat# TA336617; myelin basic protein (MBP), rat, 1:500, Abcam (Cambridge, UK), Cat# ab7349, RRID:AB_305869; Olig2, rabbit, 1:3000, ProteinTech (Planegg-Martinsried, Germany), Cat# 13999-1-AP, RRID:AB_2157541; SOX10, goat, 1:1000, R&D Systems (Wiesbaden, Germany), Cat# AF2864, RRID:AB_442208; S100, mouse, 1:5000, Thermofisher (Darmstadt, Germany), Cat# MA5-12969, RRID:AB_10983883; vesicular glutamate transporter 1 (VGluT1), guinea pig, 1:2000, Synaptic Systems (Goettingen, Germany), Cat# 135304, RRID:AB_887878.

Techniques: Labeling, Fluorescence