Article Figures & Data
Figures
- Figure 1.
Time course of loss of presynaptic sites after afferent axon cutting. A, Phase-contrast images of intact cells and a target cell with afferent elimination. Two images on the right side show regions inside orange (intact) and red (cut) squares. B, Confocal images of dendrites grown from an intact cell or a cell with afferent elimination 24 h after axon manipulation. Top images show a dendritic marker anti-MAP2 staining (green) and a dendrite-axon marker anti-neurofilament 200 (NF-H) staining (magenta). Middle images show anti-NF-H. Bottom images show anti-bassoon staining, a marker of presynaptic active zones. C, The numbers of primary dendrites 24 h after afferent elimination. D, Average fluorescence intensity of NF-H in individual images. E, Confocal images of dendrites in intact cells and cells 3 and 6 h after afferent elimination. Columns shows the anti-bassoon staining, DiI fluorescence, merged images of anti-bassoon (green) and DiI (magenta), and magnified images of regions inside yellow squares from left to right, respectively. F, Fractions of bassoon-positive spines in total spine population 3, 6, and 24 h after afferent elimination. Error bars are SEM; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant. Scale bars = 200 µm (A), 50 µm (B), 1 μm (E). Additional data about the damage to the isolated cells in afferent elimination can be found in Extended Data Figure 1-1.
- Figure 2.
Time course of changes in spines after afferent axon cutting. A, Confocal images of dendrites stained with DiI without afferent elimination (intact), or 6, 15, and 24 h after afferent elimination (cut). B, Spine density at 6, 15, 24, and 48 h after afferent elimination. C, D, Cumulative frequency of spine lengths at 6 h (C) and 24 h (D) after afferent elimination. Spines after elimination of afferent axons showed a tendency to be longer than those in intact cells at 6 h. E, Average densities of dendritic protrusions classified as long protrusion (>3 µm) in intact neurons or in neurons 6 h after afferent elimination. F, Confocal images of dendrites immunostained with PSD-95 and MAP2 antibodies without afferent elimination (intact), or 6, 15, and 24 h after afferent elimination (cut). The upper row shows images of anti-PSD-95 immunofluorescence. The lower row shows merged immunofluorescence images of anti-PSD-95 (green) and anti-MAP2 (magenta). G, Average densities of PSD-95 clusters along each dendrite at 6, 15, and 24 h after afferent elimination. Error bars are SEM; *p < 0.05, **p < 0.01, n.s. = not significant. Scale bars = 1 μm. Evaluation of damage by afferent axon elimination can be found in Extended Data Figure 2-1.
- Figure 3.
Temporal changes in PSD-95 clusters after afferent elimination. A, Confocal images of dendrites with or without afferent elimination. Images of PSD-95-GFP in the same dendrites before and 24 h after the manipulation. The pseudo color images are overlay of PSD-95-GFP (green) and DiI (magenta) 24 h after the manipulation. B, PSD-95-GFP cluster densities before and 24 h after afferent elimination. C, Time-lapse images of a PSD-95-GFP cluster moving toward a dendritic shaft. Movement of the right cluster (yellow arrowheads) became evident at 7 h 40 min after the manipulation. D, Kymographs of moving PSD-95-GFP clusters. Each image on top shows PSD-95-GFP clusters at 3 h after afferent elimination. A red line in each image on top shows the trajectory of cluster movement, along which the bottom kymograph was generated. Some PSD-95-GFP clusters split (a yellow arrow in the rightmost kymograph) and only one of the two clusters move toward the dendritic shaft. The kymographs start and end at 3 and 15 h after afferent elimination. Direction toward dendritic shafts are indicated by a red arrow. Error bars are SEM; ***p < 0.001, n.s. = not significant. Scale bars = 1 μm.
- Figure 4.
Temporal changes in PSD-95-positive spines after afferent elimination. A, Time-lapse images of dendrites with or without afferent elimination 3 and 15 h after manipulation. GFP, PSD-95-TagRFP, and their merged images (GFP; green, PSD-95-TagRFP; magenta) from left to right, respectively. Yellow arrowheads indicate loss of PSD-95-positive spines. B, Densities of PSD-95-positive spines 3 and 15 h after afferent elimination. C, Representative time-lapse sequences of a PSD-95-TagRFP cluster moving toward a dendritic shaft. The upper row shows time-lapse images of GFP. The middle row shows time-lapse images of PSD-95-TagRFP. The lower row in C is merged images of GFP (green) and PSD-95-TagRFP (magenta) with yellow arrowheads indicating translocating clusters. D, The top image shows a PSD-95-TagRFP cluster in the spine before moving. Along a red line in the image, the kymograph was generated. Each column in the kymograph shows the images of GFP, PSD-95-TagRFP or merged images of GFP (green) and PSD-95-TagRFP (magenta) from left to right, respectively. The kymographs start and end at 3 and 15 h after afferent elimination. The direction of the dendritic shaft is indicated by a red arrow. E, Representative image sequences of YFP-Homer1c (top) and PSD-95-CFP (middle) clusters moving together toward a dendritic shaft, together with an overlay of YFP-Homer1c (green) and PSD-95-CFP (magenta). Yellow arrowheads indicate a translocating cluster. F, The top image shows YFP-Homer1c and PSD-95-CFP clusters before moving. Along a red line in the image, kymographs were created. Each column in the kymographs shows YFP-Homer1c, PSD-95-CFP or overlay of YFP-Homer1c (green) and PSD-95-CFP (magenta) from left to right, respectively. The kymographs start and end at 4.5 and 15.5 h after afferent elimination. Direction toward the dendritic shaft is indicated by a red arrow. G, Time-lapse imaging of GFP-expressing dendrites with or without afferent elimination at 0 h and subsequent data acquisition at 3, 9, and 15 h. H, Density of dendritic protrusions classified as long protrusions (>4 µm in lengths) at 3 and 15 h after afferent elimination. Error bars are SEM; **p < 0.01, n.s. = not significant. Scale bars = 1 μm (A, C–F), 4 μm (G). Methods for axon-preserving alternate afferent axon elimination can be found in Extended Data Figure 4-1, and Results from alternate afferent axon elimination can be found in Extended Data Figure 4-2.
- Figure 5.
Simultaneous imaging of presynaptic and postsynaptic sites. A, Time-lapse imaging of a PSD-95-CFP cluster moving toward a dendritic shaft after disappearance of a synaptophysin-YFP cluster in contact with the PSD-95-CFP cluster. Merged images (synaptophysin-YFP; green, PSD-95-CFP; magenta) are shown in the bottom row. Yellow arrowheads indicate adjacent synaptophysin-YFP and PSD-95-CFP clusters and elimination of the synaptophysin-YFP cluster at 4 h 20 min and subsequent translocation of the PSD-95-CFP cluster starting at 4 h 30 min after afferent elimination. B, Kymographs created along the red line in the merged image at 3 h in A. Each column of the kymographs shows synaptophysin-YFP, PSD-95-CFP, and their merged images (synaptophysin-YFP; green, PSD-95-CFP; magenta) from left to right, respectively. The kymographs start and end at 3 and 15 h after afferent elimination. The direction toward the dendritic shaft is indicated by a red arrow. C, Images of a synaptophysin-YFP cluster and a PSD-95-CFP cluster at 3 h after afferent elimination with yellow ROIs set to quantitate fluorescent intensities of the synaptophysin-YFP cluster (green line in the graph) and translocation distance of the PSD-95-CFP cluster measured from the final position at 15 h after cutting (magenta line in the graph). Error bars are SEM. Scale bars = 1 μm.
- Figure 6.
Pharmacological manipulation of neuronal activity and postsynaptic function after afferent elimination. A, Confocal images of dendrites stained with DiI 24 h after afferent elimination with or without administration of either AP5, TTX, or CNQX. B, The effects of AP5, TTX, and CNQX on spine density 24 h after afferent elimination. C, Confocal images of dendrites with or without AP5 administration 24 h after afferent elimination. As a control, neurons without afferent elimination were also treated with AP5. Images in each row show anti-PSD-95 immunostaining, DiI labeling, and their merged images (PSD-95; green, DiI; magenta) from left to right. The rightmost column shows enlarged images of the areas marked by yellow squares. D, Fractions of PSD-95-positive spines in the total spine population in conditions with or without AP5 and with or without afferent elimination. Spines were imaged 24 h after afferent elimination. E, Time-lapse images of dendrites with (cut) or without (intact) afferent elimination at 3 and 15 h under AP5 administration. Afferent elimination was performed at 0 h. GFP, PSD-95-TagRFP, and their merged images (GFP; green, PSD-95-TagRFP; magenta) from left to right, respectively. F, Densities of PSD-95-positive spines at 3 and 15 h with or without afferent elimination in the presence of AP5. Error bars are SEM; *p < 0.05, **p < 0.01, n.s. = not significant. Scale bars = 1 μm. Effect of AP5 to the presynaptic sites can be found in Extended Data Figure 6-1.
Extended Data
Extended Data Figure 1-1.
Evaluation of damage to the isolated cells in afferent elimination. A, Low-magnification fluorescence images of dendrites and axons of the isolated target GFP-transfected cells before and 24 h after cutting. B, Images in the upper row show axons outside of the cutting line (the green square in A) with green arrowheads indicating disappearing axons. Images in the lower row show the soma and dendrites of the target neuron (the orange square in A) with orange arrowheads indicating the preserved dendrite. Images before, 3, 6, and 24 h after cutting are presented. C, Low-magnification images of an isolated target cell expressing GFP (green), together with phase contract image (left) or with anti-NF-H immunostaining (magenta, right). D, Higher magnification images of the same neuron in (C) with GFP (green) and anti-NF-H (magenta) fluorescence signals inside of the region marked by a yellow square in C. Yellow arrowheads indicate the axon, which starts from the target cell body and truncated at the intersection with the cutting line. Scale bars = 100 µm (A, C), 10 µm (B), 50 µm (D). Download Extended Data 1, TIF file.
Extended Data Figure 2-1
Evaluation of damage induced by afferent elimination. A, Phase-contrast and fluorescence images of a target cell with afferent elimination. The left panel shows a neuron before afferent elimination. The middle (merged image of phase contrast and PI fluorescence) and right (PI fluorescence image) panels show the neuron 6 h after cutting. PI staining detected the nuclei of the dead cells. Green arrowheads indicate injured cell bodies judged from phase-contrast images, while orange arrowheads indicate PI-positive cells. B, Phase-contrast and immunofluorescence images of a target postsynaptic neuron with (cut) or without (intact) afferent elimination. The images of the target cell 6 h after afferent elimination were presented. MAP2 (dendritic marker) fluorescence intensity outside of the circular zone (yellow) and within 250 μm from the cutting line was measured. C, Average fluorescence intensity of anti-MAP2 immunostaining was comparable between control (intact) and afferent elimination (cut). Error bars are SEM. Scale bars = 100 µm. Download Figure 2-1, TIF file.
Extended Data Figure 4-1
Afferent axon elimination with the axon from the target neuron preserved. A, B, An experimental setup of afferent axon cutting in dissociated hippocampal neuron culture. A micromanipulator-assisted cutting system on an inverted fluorescence microscopy with phase-contrast illumination (A), which enables fine movement of a needle (B, tip diameter of 0.30 mm) for precise control of the cutting trajectory on the culture surface. C, Overlay of phase-contrast and fluorescence images of a target cell (red arrow) before and after afferent elimination (yellow arrowheads). D, Lower magnification fluorescence images which include the area shown in C. Anti-bassoon immunoreactivity of the target cell (red arrows) was lower than that of neurons outside of the cutting line. Scale bars = 200 μm (C, D). Download Figure 4-1, TIF file.
Extended Data Figure 4-2
Changes in PSD-95 clusters after afferent elimination without transection of the axon growing from the target neuron. A, Confocal images of GFP (green) and PSD-95-TagRFP (magenta) with or without afferent elimination. B, Relative decrease in the density of PSD-95-positive spines 24 h after afferent elimination. C, Confocal images of GFP (green) and anti-bassoon clusters (magenta) with or without afferent elimination. D, Fractions of bassoon-positive spines in the total spine population 24 h after afferent elimination. E, A representative time-lapse sequence of two PSD-95-TagRFP clusters moving toward a dendritic shaft. The upper time-lapse sequence shows dynamics of PSD-95-TagRFP clusters. The lower time-lapse sequence is merged images of GFP (green) and PSD-95-TagRFP (magenta). White arrows indicate the first translocating cluster from spines to the dendritic shaft. Red arrows indicate the second PSD-95 cluster split into two clusters in the process of translocation. Error bars are SEM; **p < 0.01, ***p < 0.001. Scale bars = 2 μm (A, C), 1 μm (E). Download Figure 4-2, TIF file.
Extended Data Figure 6-1
Temporal changes in bassoon clusters after afferent elimination combined with AP5 administration. A, Confocal images of dendrites with bassoon and MAP2 immunofluorescence either without axon manipulation (intact), or 3 and 6 h after afferent elimination (cut). The top and third rows show anti-bassoon immunoreactivity. The second and fourth rows show double-staining with anti-bassoon (green) and anti-MAP2 (magenta). B, Average densities of bassoon clusters along dendrites 3 and 6 h after afferent elimination. Error bars are SEM; *p < 0.05. Scale bars = 1 μm. Download Figure 6-1, TIF file.
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