Territories of heterologous inputs onto Purkinje cell dendrites are segregated by mGluR1-dependent parallel fiber synapse elimination

Abstract

In Purkinje cells (PCs) of the cerebellum, a single "winner" climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9-15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.

Keywords: Purkinje cell; cerebellum; climbing fiber; parallel fiber synapse elimination.

Fig. 1.

Development of CF and PF projections onto PC dendrites in postnatal mouse cerebella. (A–F) Triple fluorescent labeling for CFs (red, BDA tracer labeling), PF terminals (blue, VGluT1 immunofluorescence), and PCs (green, calbindin immunofluorescence) at P7 (A), P9 (B), P12 (C), P15 (D), P20 (E), and P30 (F). Arrowheads indicate the tip of the CF projection, and arrows indicate the somatodendritic border of PCs. (Scale bars, 10 µm.) (G) The mean vertical height (µm, mean ± SD, n = 3 mice at each stage) to the tips of PC dendrites (green) or CFs (yellow). To evaluate the vertical height, 10 PC dendrites and 10 CFs were analyzed in each mouse. (H) The vertical height of the CF projection relative to that of PC dendrites (%, mean ± SD, n = 3 mice at each stage).

Fig. S1.

(A–F) Bright-field micrographs of BDA-labeled CFs used for ultrastructural analysis from P7 to P30. For ultrastructural reconstruction, we selected BDA-labeled CFs whose trajectories could be traced from PC somata to the tips of CFs. Red and blue arrows indicate approximate portions of the most distal CF synapses (CFd) and the most proximal parallel fiber synapses (PFp) shown in G–X. After taking these micrographs, sections were subjected to pre-embedding silver-enhanced immunogold microscopy for VGluT1 to label PF terminals, and embedded in Epon812. Asterisks indicate PC somata. (G–X) Electron micrographs showing the most distal CF synapses and the most proximal PF synapses at P7 (G and H), P9 (I and J), P12 (K and L), P15 (M–P), P20 (Q–T), and P30 (U–X). Boxed regions in G–L are enlarged in Insets, whereas those in M, N, Q, R, U, and V are enlarged in O, P, S, T, W, and X, respectively. Insets in G, I, and K and closer views of O, S, and W show the most distal CF synapses (CFd) labeled with BDA tracer (diffuse DAB precipitates). Insets in H, J, and L and closer views of P, T, and X show the most proximal PF synapses (PFp) labeled for VGluT1 (metal particles). Arrows indicate the coexistence of CFd with PF synapses or of PFp with CF synapses (CF). Pairs of arrowheads indicate both edges of the postsynaptic density. PC dendrites or somata are pseudocolored in green. Note reduced calibers of CFd- and PFp-associated PC dendrites because of distal elongation of CF territory or distal retraction of PF territory, respectively, during early postnatal period. Also note that the most distal CF synapses at P7 are observed at apical portions of PC somata or somatodendritic border. (Scale bars, 10 μm in A–F; 500 nm in G–X.)

Fig. 2.

Reconstructed dendritic innervation in wild-type PCs at P7 (A), P9 (B and G), P12 (C and H), P15 (D and I), P20 (E and J), and P30 (F and K). (A–F) CF synapses (yellow circles on red lines) and PF synapses (blue circle) are shown to the right of PC somata and dendrites (gray), whereas GABAergic spine-type synapses (green circle) and free spines (black square) are to the left of dendrites. One of the three PCs examined at each stage is illustrated here, with the rest being shown in Fig. S3. (G–K) Three-dimensional reconstructed images of dendritic innervation. Dendritic portions of double-headed arrows in B–F are illustrated.

Fig. S2.

Electron micrographs showing synapses and free spines on PC dendrites at P15. (A) CF synapse on a PC spine (Sp), identified by diffuse dark precipitates for the anterograde tracer BDA in the presynaptic terminal and an asymmetrical contact characterized by a thick postsynaptic density (arrowheads). (B) PF synapses on PC spines, identified by metal particle labeling for VGluT1 and asymmetrical contacts (arrowheads). (C) GABAergic synapse on a dendritic shaft of PC, identified by metal particle labeling for VIAAT and a symmetrical contact (arrowheads). (D) Symmetrical synapse on a right PC spine. (E) GABAergic synapse on a PC spine, identified by metal particle labeling for VIAAT and a symmetrical contact (arrowheads). (F) Serial images of a free spine, which is surrounded by Bergmann glia and lacking synaptic contact. (Scale bars, 500 nm.)

Fig. S3.

Reconstructed dendritic innervation in wild-type PCs at P7 (A), P9 (B), P12 (C), P15 (D), P20 (E), and P30 (F). CF synapses (yellow circles on red lines) and PF synapses (blue circle) are shown to the right of PC somata and dendrites (gray), whereas GABAergic spine-type synapses (green circle) and free spines (black square) are to the left of dendrites. See also Fig. 2 A–F.

Fig. 3.

Developmental changes in territories and patterns of CF and PF innervation on PC dendrites. (A) Schematic drawings showing the mean path length of the three dendritic domains at P7–P30 (µm, mean ± SD). Shown are the CF-dominant PCD-I (orange), the overlapping PCD-II (pink), and the PF-dominant PCD-III (blue) domains. (B) The mean path length of CF territories (yellow columns) and PF territories (blue columns) (µm, mean ± SD, n = 3 dendrites each). From P15 onwards, the CF territory elongates, whereas the PF territory is reciprocally shortened. (C–F) Lentivirus-mediated GFP labeling of PCs at P15 (C and D) and P20 (E and F). Boxed regions in C and E are enlarged in D and F, respectively. Arrowheads indicate spiny protrusions from proximal shaft dendrites. (Scale bars, 5 μm.) (G) The mean number of CF (yellow bars) and PF (blue bars) synapses per analyzed dendritic track (mean ± SD, n = 3 dendrites at each stage). (H) The mean density of CF synapses in the CF territory (yellow bars) and PF synapses in the PF territory (blue bars) (mean ± SD, n = 3). Synapse density is indicated per 1 µm of dendritic path length. (I) The mean number of free spines per analyzed dendritic track (n = 3, mean ± SD). The P value was calculated using Student’s t test. *P < 0.05; **P < 0.01.

Fig. S4.

Schematic showing how the path length of curving and leaning dendrites was calculated. Assuming that dendrites are cylindrical, the red triangle defined by the long (D_L) and short (D_S) diameters of dendritic profiles is similar in shape to the yellow triangle defined by the path length (L) and section thickness (t). Therefore, the path length in each section is calculated as D_L/D_S × t, and is summed to obtain the total path length of the CF or PF territory in a given PC.

Fig. S5.

Electrophysiological analysis of mEPSCs in PCs. (A) Sample traces of mEPSCs at P15 (Upper) and P20 (Lower). Representative events (red arrow heads) are shown in higher time and current resolutions on the right. (B) Normalized frequency distribution histograms for the 10–90% rise times of mEPSCs sampled at P14–P16 (blue, n = 31) and at P18–P20 (pink, n = 23). The normalized frequency of mEPSCs was higher at P14–P16 than at P18–P20 for the rise times of 0.4-, 0.6-, and 2.6-ms bins, and vice versa for 1.8-ms bin (P < 0.05, t test). In the following analyses, we focused on the events with the rise times of 0.2–0.6 ms that are considered to arise from synapses on the proximal portions of PC dendrites. A rationale for this notion is that the rise times of quantal EPSCs arising from CFs, which form synapses on proximal PC dendrite, were shorter than 1 ms for most of the events (see E). (C) Incidence of mEPSCs with rise times of 0.2–0.6 ms at P14–P16 was significantly higher than that at P18–P20 (t test, P = 0.00844). (D) Asynchronous quantal EPSCs arising from CFs recorded in the external solution containing Sr²⁺ (1 mM) from PCs at P14–P16. (Left) Representative traces of EPSCs. Events were sampled during a 500-ms period after the CF stimulation. The red arrow indicates time points when CFs were stimulated. Representative events (red arrowhead) are shown in higher time and current resolutions on the right. (Right) Normalized frequency distribution histograms for the 10–90% rise times of quantal EPSCs at P14–P16 (n = 10). Note that the rise time was faster than 1 ms for most of the events. (E) Normalized frequency distribution histograms for the 10–90% rise times of mEPSCs in the absence (open circles) and the presence (filled circles) of l-AP4 (50 µM) at P14–P16 (Upper, n = 8) and at P18–P20 (Lower, n= 5). Bar graphs on the right of the histograms show the incidence of mEPSCs with rise times of 0.2–0.6 ms in the absence (open circles) and the presence (filled circles) of l-AP4 (50 µM). Note that l-AP4 significantly reduced the incidence at P14–P16 (Upper, paired-t test, n = 8, P = 0.0044) but not at P18–P20 (Lower, n = 5, P = 0.08). Histograms for l-AP4 treatment are normalized to the number of events elicited in the control solution. (F) Incidence of quantal EPSCs arising from CFs was not affected by l-AP4 (50 µM) but markedly suppressed by the group II mGluR agonist DCG IV (1 µM) that are known to suppress CF-mediated EPSCs (50). (One-way ANOVA with Holm–Sidak post hoc test, control n = 8, l-AP4 n = 8, DCG IV n = 6, P < 0.001). Three sample traces (Left) and summary bar graph (Right) showing the effects of l-AP4 (50 µM) and DCG-IV (1 µM). Red arrows indicate time points when CFs were stimulated. For B, C, D, E, and F, values are expressed as mean ± SEM *P < 0.05, **P < 0.01.

Fig. S6.

CF and PF innervation in mGluR1-KO (Left) and PKCγ-KO (Right) mice. (A–D) Triple fluorescent labeling for CFs (red, BDA tracer labeling), PF terminals (blue, VGluT1 immunofluorescence), and PCs (green, calbindin immunofluorescence) at P15 (A and C) and P30 (B and D) in mGluR1-KO (A and B) and PKCγ-KO (C and D) mice. Arrowheads indicate the tip of the CF projection, whereas arrows indicate the somatodendritic border of PCs. (Scale bars, 10 μm.) (E–H) Reconstructed dendritic innervation in three PCs at P15 (E and G) and P30 (F and H) in mGluR1-KO (E and F) and PKCγ-KO (G and H) mice. See legends for Figs. 1 A–F and 2 A–F.

Fig. 4.

Arrested refinement of CF and PF innervation in mGluR1-KO and PKCγ-KO mice. (A) Schematic drawings showing the mean path length of three dendritic domains (µm, mean ± SD). See legend in Fig. 3A. (B) The mean path length of CF and PF territories. Note a marked shortening of the CF territory with a reciprocal elongation of the PF territory in both mutants at P30. (C and D) The mean number (C) and density (D) of CF (yellow columns) and PF (blue columns) synapses (mean ± SD, n = 3 dendrites at each stage). Data from wild-type mice at P15 and P30 are reproduced from Fig. 3A. Statistical differences between mutant and wild-type mice are indicated with asterisks (*P < 0.05; **P < 0.01).

Fig. 5.

Restored refinement in CF and PF innervation after lentiviral transfection of mGluR1α in mGluR1-KO PCs. (A–F) Triple fluorescent labeling for GFP (red), mGluR1α (blue), and calbindin (green) in mGluR1α/GFP-transfected mGluR1-KO cerebellum at P40. Untransfected (A, C, and D) and transfected (B, E, and F) cerebellar portions are shown. (G and H) Reconstructed dendritic innervation in mGluR1-KO (G) and rescued (H) PCs. One of the three PCs examined in mGluR1-KO and mGluR1α-rescued PCs is illustrated here, with the rest being shown in Fig. S7. (I) The mean path lengths of the three dendritic domains (µm, mean ± SD). (J–L) Bar graphs showing the mean path lengths of CF and PF territories (J), the mean number of CF synapses, PF synapses, and free spines (K), and the mean density of CF and PF synapses (L). Note the emergence of free spines in the CF territory of rescued PCs (black squares in H and gray column in K). See legends for Figs. 2 and 3. (Scale bars, 100 µm in A and B; 10 µm in C–F.)

Fig. S7.

Reconstructed dendritic innervation in mGluR1-KO (A) and mGluR1α-rescued (B) PCs. See also Fig. 5 H and I.

Fig. 6.

Territory refinement by CF and PF innervation in PCs. In the territory overlap phase (P9–P15), a winner CF (red) translocates to PC dendrites, weaker CFs (pink) remain on the soma, and newly differentiated PF synapses (blue) are added on to the growing tips of dendritic trees. All of these events fuel synaptic competitions among homologous and heterologous inputs, but their territories of innervation do not remain segregated. In the territory segregation phase (P15 onward), PF synapses are eliminated from overlapping dendritic portions, leading to the segregation of the CF and PF territories. Moreover, redundant CF synapses remaining on PC somata are eliminated, establishing CF monoinnervation. The mGluR1 signaling pathway promotes both the elimination of CF synapses from the soma (the late phase of CF synapse elimination, P12–P17) and the elimination of PF synapses from proximal dendrites. As a result, redundant innervations by homologous and heterologous inputs are refined into input-selective wiring in PCs (i.e., CF monoinnervation and segregated territories of CF and PF innervation).