Dendritic translocation establishes the winner in cerebellar climbing fiber synapse elimination

Abstract

In many regions of the developing mammalian nervous system, functional synaptic circuitry is formed by competitive elimination of early formed redundant synapses. However, how winning synapses emerge through competition remains unclear in the brain largely because of the technical difficulty of directly observing this dynamic cellular process in vivo. Here, we developed a method of two-photon multicolor vital imaging to observe competitive elimination of supernumerary climbing fibers (CFs) in the cerebellum of live mouse pups. At birth, each Purkinje cell (PC) in the cerebellar cortex is innervated by multiple CFs; an activity-dependent regression of supernumerary CFs ultimately yields a single innervation for most PCs by postnatal day 21. As supernumerary CFs are pruned, the terminal field of CFs translocates from the soma to the dendrites of PCs. In vivo time-lapse imaging of CF elimination revealed that (1) CF terminals were highly motile on the soma, but their motility was significantly reduced on dendrites; (2) only one CF could translocate to the dendrites whereas their competitors were restricted to perisomatic regions; and (3) the CF that began dendritic translocation became the winner. Moreover, selective photo-ablation of the winning CF (that undergoes dendritic translocation) reversed the fate of its losing competitor. These results indicate that dendritic translocation is a key cellular event that determines the winner during CF elimination. We propose that CF terminals are selectively stabilized on dendrites, providing irreversible competitive vigor to the first CF to form dendritic synapses.

Figure 1.

Time-lapse in vivo imaging of CF dendritic translocation. A, A mouse pup (P10) 3 d after the surgery. An arrow indicates the small rectangular shaped window. An arrowhead indicates the metal plate used to hold the head still in the anesthetized animals. B, A higher magnification view of the cranial window shown in A. C, The same CF innervating a single PC repeatedly imaged in vivo by two-photon microscopy during the period of late phase CF elimination. CF was labeled by TMR-dextran. Top, Maximum projections showing top-down views of the CF. Bottom: Sagittal views of the same CF reconstructed from the same image stacks collected for the top panels. A whole CF tree (i.e., a terminal portion of olivocerebellar axon between the PC layer and pial surface) is shown in both the top-down and sagittal views. Asterisks indicate approximate location of the PC soma innervated by the CF. Arrowheads indicate corresponding points on the dendrites in the top and the bottom panels. In the bottom panels, dashed lines indicate approximate location of the PC layer and pial surface is located at the right side of the images (hence, the CF extends toward right of the images as the dendritic translocation proceeds). Scale bars: 10 μm. Note that (1) the scale differs between the top and the bottom panels and (2) the primary dendrites of the PC innervated by the CF were not entirely parallel to the optical axis. Therefore, in the top panels, the CF appears to extend away from the soma (toward top of the images) as the dendritic translocation proceeds. The CF began dendritic translocation at P11 (i.e., extended its arbor toward right in the bottom panels). At P13, the CF innervated both the soma and dendrites of the PC. The somatic innervation became less prominent afterward (see the image at P16) and mostly disappeared by P20, whereas the dendritic innervation persisted. D, Fixed cerebellar slices of nefl-EGFP tg mice (P19) were immunostained with anti-GFP (green) and anti-calbindin-D-28K antibody (a marker for PC; red). Note: CFs are the only GFP-positive structures in the cerebellar molecular layer. Scale bar, 10 μm. E, Fixed slices of inferior olive derived from nefl-EGFP tg mice (P12) were immunostained with anti-GFP (green) and anti-NeuN antibody (a neuronal marker; red). Note: Most olivary neurons are GFP positive. Scale bar, 20 μm. F, Time-lapse in vivo imaging performed with an nefl-EGFP tg mouse (without tracer injection). Maximum projections showing top-down views of the CFs are shown. The same imaging field was approximately located by approximate location in the cranial window and then confirmed by low-magnification two-photon images. As shown in this example, the spatial pattern of EGFP-positive CFs is a reliable landmark to locate the same set of CFs for time-lapse imaging. These projections are color coded such that optical planes above PC soma are red and others (planes below and at PC soma) are green. Therefore, red color indicates the CF portions translocating to the dendrites. Scale bar, 20 μm.

Figure 2.

Dendritic CF terminals are more stable than somatic terminals. A, Time-lapse images of CFs labeled by EGFP in a nefl-EGFP tg mouse. CF varicosities located on the soma and primary dendrite of the same PC are compared. Maximum projections containing most of the somatic varicosities (top two images) and the 3 μm segment of the proximal primary dendrite (bottom two images) ∼8–10 μm above the soma are shown. Scale bar, 10 μm. B, The fraction of varicosities that appeared or disappeared over 1 d was compared between soma and dendrites in each PC. A pair of open circles connected by a line represents the data from a single PC. Horizontal bars represent the average of eight analyzed PCs. Asterisk denotes p < 0.01 (χ² test). Note that each PC is innervated by a single CF in >50% of the cases. Hence, these data indicate that even the varicosities of single CFs are more stable on the dendrites than the soma. C, Fixed cerebellar slices of nefl-EGFP tg mice (P13) were immunostained with anti-GFP and anti-VGluT2 antibody. White dashed lines indicate the approximate location of the border between the soma and proximal dendrites. Most EGFP-positive CF varicosities were also VGluT2 positive on both the soma and dendrites, indicating that they are functional terminals. Scale bar, 10 μm.

Figure 3.

Multicolor in vivo imaging of two CFs competing for a same PC. A–C, The left PC (asterisks) is innervated by both a EGFP single-positive CF (arrowheads in A and C) and a EGFP/TMR double-positive CF (arrows in A–C) at P9. The middle and right PCs are innervated by only EGFP single-positive CFs. Single Z-sections (not maximum projections) are shown here for validating the multicolor labeling. Scale bar, 10 μm. D, Two colors are mostly sufficient to distinguish all individual CFs competing for the same PC during late phase CF elimination. Left, Representative traces of CF-EPSCs innervated by single (top), double (middle), and triple (bottom) CFs. CF-EPSCs, evoked by gradually increased stimulus intensity, were overlaid. Holding potential was −20 mV. Right, A histogram showing fractions of PCs innervated by single, double, and tripe CFs. Approximately 90% of PCs were innervated by single or double CFs at P11–P12.

Figure 4.

Time-lapse multicolor in vivo imaging of the same set of competing CFs. CFs contain either EGFP only (green) or EGFP/TMR (yellow or orange). A–D, Top (A1–D1), Maximum projections showing top-down views of CFs that involve both somatic and dendritic regions. Asterisks indicate PCs innervated by both green and yellow CFs. White circles indicate neighboring PCs. Bottom (A2–D2), Sagittal views of the same multiply innervated PCs (indicated by asterisks in A1–D1) reconstructed from the image stacks collected for the top parts. Whole CF trees (i.e., terminal portion of olivocerebellar axons between the PC layer and pial surface) are shown. White dashed lines indicate approximate location of the border between soma and proximal dendrites. Pial surface is located at the right side of the images. Arrowheads indicate corresponding points on the dendrites in the top and bottom. Since two adjacent PCs were innervated by multiple CFs in B, arrows (for the top left PC) are used in addition to arrowheads (for the bottom right PC). In the case of A and C, the green CF was the winner and translocated. In B, the yellow CF was the winner for the top left PC whereas the green CF was the winner for the bottom right PC. In D, a green CF was dominant at P10, but an orange CF dominated the green CF later and became the winner (flip-flop). E, A rare example of dual-CF dendritic translocation found in one PC. Maximum projections (E1) and sagittal views (E2) are shown as explained above for A–D, except that (1) only a part of the CF tree (soma and proximal portion of dendrites) are shown for P21, (2) arrowheads indicate the dendritic portion of the green CF, and (3) an arrow indicates somatic varicosities of the green CF at P21. Note that the green CF first translocated to the dendrites, but the yellow CF later translocated as well. Although the yellow CF eventually innervated most of the dendritic surfaces, the green CF still remained on dendrites (arrowhead) and soma (arrow) even at P21. Scale bars: 10 μm in all images. F, The height of CFs in the molecular layer was quantified and compared between winner and loser at each time point. Error bars indicate SEM. **p < 0.001 (Mann–Whitney U test). Note that these measurements may not be as accurate as conventional histological analyses because of the poor axial resolution of two-photon microscopy and brain pulsation during image acquisition.

Figure 5.

Functional analysis of morphologically identified CFs. A, A multicolor image of two competing CFs in an acute cerebellar slice. The orange and green CFs (arrowheads) innervate a PC (asterisk) before dendritic translocation. The green CF contains only EGFP and the orange CF contains a calcium indicator (OGB1) in addition to the TMR and EGFP in the previous figures. Scale bar, 10 μm. B, CF-EPSCs were evoked by gradually increasing the stimulus intensity to CF axons in the granule cell layer. Traces are overlaid. C, Changes of green fluorescence upon CF stimulation were measured in the dashed box in A, simultaneously with the CF-EPSCs recordings. Traces were averaged three times. Arrow indicates the timing when the stimulation was given. D, Amplitude of EPSCs (top) and Ca²⁺ transient (bottom) as a function of stimulus intensity. The first EPSC step (red traces in B) shared the same threshold as the Ca²⁺ signal, indicating that it was evoked by the orange CF (containing Ca²⁺ indicator). The second EPSC step was not accompanied by an increase in the Ca²⁺ signal because this step was the green CF (not containing Ca²⁺ indicator). E, Relative synaptic strength of TMR/OGB1/EGFP triple-positive CFs (a ratio of the first EPSC amplitude to the total EPSC amplitude) is plotted against their relative innervation area (a ratio of somatic area innervated by the TMR/OGB1/EGFP triple-positive CF to the both CFs). Morphological dominance of a CF correlates with its functional dominance in white quadrants, whereas they are not correlated in gray quadrants. Note: Data obtained from all six PCs analyzed are in the white quadrants. F, The distribution of the eventual winner CFs as a function of their relative innervation area on the somata. Multicolor in vivo time-lapse images taken before dendritic translocation proceeded were analyzed. Of 35 winner CFs, the relative innervation area of 10 CFs was <50% (a dashed line in the histogram) on the somata before translocation, suggesting that flip-flop occurred.

Figure 6.

Selective photo-ablation of the “winning” CF (i.e., translocating onto the dendrite) causes the “losing CF” (i.e., nontranslocating) to translocate. A, A594 can be selectively overexcited with little excitation of EGFP at 830 nm. CFs contain either EGFP only or A594/EGFP. Asterisks indicate a PC innervated by the A594/EGFP double-positive CF. Green and red channels are individually shown. Left, CFs were excited at 830 nm with 30 mW of average laser power. Right, The same field of view was excited at 890 nm with 75 mW of average laser power. The detector setting was identical between green and red channels and the setting was unchanged between 830 and 890 nm excitation. B, Overexcitation of A594 at 830 nm enabled selective photo-ablation of A594-containing CFs in vivo. CFs contain either EGFP only (green) or A594/EGFP/TMR (yellow). Only one CF in this image is A594/EGFP/TMR triple positive. All images were taken by multicolor excitation at 870–890 nm. Top, Maximum projections showing top-down views of the CFs. At P8, a PC (asterisk) was innervated by both green and yellow CFs but the yellow CF was dominant. After taking the image, 830 nm laser light was delivered to the entire imaging field for selective overexcitation of A594. Two days later (P10), the yellow CF lost fine structural details and had several unusually big swellings: beaded structures that are typical for damaged neurites. The yellow CF disappeared at P11. Bottom, P8 and P10 show higher magnification views of red and green channels. P11 and P12 show sagittal views of the PC (asterisk) innervated by the yellow CF until P10. A green CF that co-innervated the PC was not ablated and showed developmental growth, including growth onto dendrites obvious in the P12 sagittal view. Note: EGFP signals near the bottom of the P12 image (top) are weak because of slight bone regeneration under the cranial window. C, D, Selective photo-ablation of the winning (translocating) CFs. CFs contain either EGFP only (green) or A594/EGFP/TMR (yellow or orange). All images were taken by multicolor excitation at 870–890 nm. C1, D1, Maximum projections showing top-down views of the CFs. At P11, PCs (asterisks) were innervated by both yellow/orange (winning) and green (losing) CFs. Neighboring PCs are indicated by white circles. C2, D2, Sagittal views of the same multiply innervated PCs (indicated by asterisks in C1 and D1) reconstructed from the image stacks collected for C1 and D1, respectively. Whole CF trees (i.e., terminal portion of olivocerebellar axons between the PC layer and pial surface) are shown. White dashed lines indicate approximate location of the border between the soma and proximal dendrite. Pial surface is located at the right side of the images. Arrowheads indicate corresponding points on the dendrites in the top-down views (C1, D1) and sagittal views (C2, D2). Scale bars: 10 μm in all images. Note: The yellow/orange CFs were already translocating to the dendrites at P11. Selective photo-ablation of these yellow/orange CFs was performed after taking the images at P11. In C, the yellow CF lost fine structural details at P13 and had several big swellings: beaded structures that are typical for damaged neurites. In contrast, the green CF expanded its territory on the somata and later translocated to the dendrite (P15 and 17). In D, the orange CF disappeared at P13. The green CF slowly expanded its territory on the somata and later translocated to the dendrite (P19). E, The height of CFs in the molecular layer was quantified and compared between the ablated CF (presumable winner under normal circumstances) and its nonablated competitor. The left and right side of the dashed line indicate before and after photo-ablation, respectively. Error bars indicate SEM. Asterisks and “ns” denote p < 0.01 and p > 0.05, respectively (Mann–Whitney U test). Note that, immediately after photo-ablation (P13–P14), the difference between ablated and nonablated CFs was not significant, because 4 of 7 nonablated CFs had not yet begun dendritic translocation.