Article Figures & Data
Figures
- Figure 1.
Depletion of microglia with PLX5622. A, Timeline of PLX5622 administration. PLX5622 was intraperitoneally injected into pregnant mothers once a day from E12 and to the pups once a day from P0. Animals were analyzed at P0, P4, and P6 based on previous studies (Fujimoto et al., 2023). B, Distribution of microglia in the olfactory bulb of control (top) and PLX5622-treated (bottom) mice. Anti-Iba1 staining shows microglia. GL, glomerular layer; MCL, mitral cell layer. Scale bars are 300 and 50 μm. D, dorsal; V, ventral; M, medial; L, lateral. C, Quantification of Iba1-positive microglia in the olfactory bulb. One olfactory bulb per animal was analyzed. Every three sections (i.e., every 48 μm) were analyzed for each olfactory bulb for quantification (28–56 sections of 16-μm thickness per animal). Data are from three mice each. ***p < 0.001, **p < 0.01 (Welch’s t test). See also Extended Data Figures 1-1 and 1-2 for pilot experiments with PLX3397.
- Figure 2.
Microglial depletion does not affect dendritic pruning of mitral cells. A, Representative images and reconstructions of mitral cells in control and PLX5622-treated mice at P4 and P6. White and black arrowheads indicate somata and primary dendrites of mitral cells, respectively. Scale bars represent 50 μm. B, Quantification of the number of glomeruli innervated by single mitral cells. C, Quantification of lateral dendrite formation. In this quantification, we excluded mitral cells whose lateral dendrites were densely labeled and difficult to trace. N.S., nonsignificant (χ2 test compared with the control). Number of neurons (n) are indicated in parentheses. Data are from five or six mice per group.
- Figure 3.
Microglial depletion does not affect the formation of excitatory synapses in mitral cells. A, Representative images of dendritic tufts and PSDΔ1.2-EGFP puncta (excitatory synapses) of mitral cells in the control and PLX5622-treated mice at P4 and P6. Images are from glomerular layer. Scale bars represent 10 μm. B, C, Quantification of dendritic tuft volumes, number of PSD puncta, PSD puncta density, and PSD size at P4 (B) and P6 (C). N.S., nonsignificant (Welch’s t test). Number of neurons (n) are indicated in parentheses. Data are from five or six mice per group.
- Figure 4.
Microglial depletion does not affect axonal projection of OSNs. A, Labeling a subset of OSN axons with DBA lectin. Numbers of OMP-positive and DBA-positive (white arrows) glomeruli were quantified. Scale bars are 300 μm (left) and 50 μm (right). D, dorsal; V, ventral; M, medial; L, lateral. B, OSN projection in control (top) and PLX5622-treated (bottom) mice. Scale bars are 300 μm (left) and 100 μm (right). C–E, Numbers of DBA-positive glomeruli (C), OMP-positive glomeruli (D), and DBA/OMP ratio (E). One olfactory bulb (hemibrain) per animal was analyzed for quantification every three sections (i.e., sampled every 48 μm). N.S., nonsignificant (Welch’s t test). Number of animals (N) are shown in parentheses.
- Figure 5.
Microglial depletion increases the number of excitatory synapses in the olfactory bulb granule cells. A, PSDΔ1.2-EGFP puncta in the granule cells in the olfactory bulb of control and PLX5622-treated mice. PSDΔ1.2-EGFP was introduced to a subset of granule cells using in utero electroporation at E13. Scale bars represent 50 μm (left), 10 μm (middle), and 5 μm (right). B, Quantification of PSDΔ1.2-EGFP puncta in granule cells. **p < 0.01, N.S., nonsignificant (Welch’s t test). Number of neurons (n) are shown in parentheses. Data are from three or four mice per group. C, Excitatory synapses were visualized with anti-Homer1 antibody. Olfactory bulb sections of control (top) and PLX5622-treated (bottom) mice are shown. Scale bars represent 50 μm (left), 15 μm (right top), and 5 μm (right bottom). GL, glomerular layer; MCL, mitral cell layer. D, Quantification of Homer1+ puncta density in the external plexiform layer of the olfactory bulb per volume. The density of Homer1+ puncta was unchanged at P0 and P4 but was significantly increased at P6. *p < 0.05 (Welch’s t test). Number of animals (N) are shown in parentheses.
- Figure 6.
Microglial depletion does not affect the dendrite pruning of L4 neurons in the barrel cortex. A, Representative dendritic reconstruction of L4 neurons in the barrel cortex. Barrel hollow (green or dark gray) was identified by anti-VGluT2 immunostaining. White arrowheads indicate somata of analyzed L4 neurons. Scale bars are 150 μm (top) and 50 μm (middle and bottom). B, Dendritic orientation. The x-axis shows the percentage of the dendritic length found within the major follow. The y-axis shows the number of neurons. C, Quantifications of dendritic orientation. Mean ratios (%) of dendritic length in the major follow (green), septa (light gray), and adjacent hollows (dark gray) are shown. N.S., nonsignificant (Welch’s t test). D, Quantification of total dendritic length. N.S., nonsignificant (Welch’s t test). E, Quantification of the number of primary branches. N.S., nonsignificant (Welch’s t test). Number of neurons (n) are shown in parentheses. Data are from three mice per group.
Extended Data
Extended Data Figure 1-1
Microglial depletion by postnatal PLX3397 treatment. A, Timeline of PLX3397 treatment in postnatal mice. PLX3397 was intraperitoneally injected to pups twice a day from P0 to P6. Mice were analyzed at P0, P4, and P6. B, Relative number of microglia at P6 after treatment with different doses of PLX3397. The x-axis shows the dose of PLX3397 injection (mg/kg body weight). ***p < 0.001, **p < 0.01, *p < 0.05 (Welch’s t test, compared with the control). C, Anti-Iba1 staining in the olfactory bulb of control and PLX3397-treated (20 mg/kg body weight) mice. Scale bars represent 300 μm (left) and 50 μm (right). D, Relative number of microglia at different stages. ***p < 0.001, *p < 0.05 (Welch’s t test, compared with the control). E, Representative traces of mitral cells in control and PLX3397-treated mice. F, Quantification of glomeruli innervated by individual mitral cells. N.S., nonsignificant (χ2 test compared to the control). Number of neurons (n) are indicated in parentheses. Data are from three or four mice per group. Download Figure 1-1, TIF file.
Extended Data Figure 1-2
Microglial depletion by PLX3397 treatment from embryonic stages. A, Timeline of PLX3397 treatment from embryonic stages. PLX3397 was intraperitoneally injected into the pregnant mother twice a day from E6 and pups after birth. Mice were analyzed at P0, P4, and P6. B, Anti-Iba1 staining in the olfactory bulb of control and PLX3397 (20 mg/kg body weight) treated mice. Scale bars represent 300 μm (left) and 50 μm (right). C, Relative number of microglia at different stages. ***p < 0.001, **p < 0.01, *p < 0.05 (Welch’s t test, compared with the control). D, Representative traces of mitral cells in control and PLX3397-treated mice. E, Quantification of glomeruli innervated by individual mitral cells. N.S., nonsignificant (χ2 test compared to the control). Number of neurons (n) are indicated in parentheses. Data are from three mice per group. Download Figure 1-2, TIF file.
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