Developmental switching of perisomatic innervation from climbing fibers to basket cell fibers in cerebellar Purkinje cells

Abstract

In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs was most developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABA(A) receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABA(A) receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals.

Figure 1.

A₁–E₃, Developmental profile of CF and BF projection to PC somata at P7 (A₁–A₃), P9 (B₁–B₃), P12 (C₁–C₃), P15 (D₁–D₃), and P20 (E₁–E₃). Triple fluorescent labeling for CFs with BDA (red, A₂–E₂, A₃–E₃), for inhibitory terminals with VIAAT antibody (green, A₁–E₁, A₃–E₃), and for PCs with calbindin antibody (blue, A₁–E₃) is shown. Asterisks indicate PC somata. Double and single arrowheads indicate the tip and trajectory of CF projection, respectively. Large arrowheads in E indicate the pinceau formation surrounding the axon initial segment of PCs. Scale bars: 20 μm.

Figure 2.

Six types of perisomatic synapses and free spines in PCs at P7. A–J, CF–spine synapse (A), BF–spine synapse (B, C), BF–soma synapse (D, E), other somatic synapse (F), FS–BF (G, H), and free spines surrounded by BGs (FS–BG; I, J). In A, the CF is labeled with dark filling of BDA-mediated DAB precipitates. In C and E, BFs are labeled by immunogold for VIAAT. Note that synaptic contact in B is a symmetrical type, whereas that in C is an asymmetrical type. From the dense filling of synaptic vesicles and symmetrical contact, the synapse in F is judged as a recurrent PC synapse. Arrowheads indicate the edges of synaptic contact. Adjacent images of boxed regions in G and I are shown in H1–H4 and J1–J4, respectively. PCS, Purkinje cell soma; r-PC, recurrent Purkinje cell axon. Scale bars: 0.5 μm.

Figure 3.

Histograms showing developmental changes in the density and composition of perisomatic synapses and free spines. A, The density of the total perisomatic synapses. B, The density of the total somatic spines. C–H, The density of the CF–spine synapse (C), BF–spine synapse (D), BF–soma synapse (E), other somatic synapse (F), free spines surrounded by BFs (G), and free spines surrounded by BGs (H). I, The composition of perisomatic synapses. The density is expressed as the number per 100 μm² of the somatic surface area. Numerals to right of error bars in A–H indicate the mean ± SD, while those in I indicate the percentage of each perisomatic synapse (n = 3 mice for each).

Figure 4.

Horizontal electron micrographs through PC somata. A, P7. B, P9. C, P12. D, P15. E, P20. PCs are pseudocolored in blue, BDA-labeled CFs are in brown (also indicated by arrows), and BF terminals are in green (asterisks). Boxed regions in A–E are enlarged in Figure 5A–E, respectively. Scale bars: 5 μm.

Figure 5.

A–J, Electron micrographs (A–E) and reconstructed illustrations (F–J) of perisomatic synapses and free spines in PCs. A, F, P7. B, G, P9. C, H, P12. D, I, P15. E, J, P20. A–E are enlarged views of boxed regions in Fig. 4. F–J, Illustrations reconstructed from serial sections including A–E. BDA-labeled CFs, BFs, and PCs are pseudocolored in red, green, and blue, respectively. Arrowheads indicate the edge of synaptic contact. BF–Sp, BF–spine synapse; BF–Sm, BF–soma synapse; CF-Sp, CF–spine synapse; BF–BG, free spine surrounded by Bergmann glia; PCS, Purkinje cell soma; PSD-I, thick PSD forming an asymmetrical type of contact. Scale bars: 1 μm.

Figure 6.

Schematic illustrations showing the distribution of perisomatic synapses and free spines. A, P7; B, P9. C, P12. D, P15. E, P20. Four types of perisomatic synapses and two types of free spines are plotted on flattened somatic surface of three reconstructed PCs.

Figure 7.

Specificity of GABA_ARα1 and gephyrin antibodies. A, Immunoblot with GABA_ARα1 antibody using HEK cell lysates transfected with plasmid vector encoding GABA_ARα1 (left) or GABA_ARα2 cDNA (middle) and using the PSD fraction of adult mouse brains (right). B, C, Immunoblot with two gephyrin antibodies using HEK cell lysates transfected with plasmid vector encoding gephyrin cDNA (left) or plasmid vector only (middle) and using the PSD fraction of adult mouse brains (right). D–F, Immunofluorescence with GABA_ARα1 (D), gephyrin (1–45 aa residues; E), and gephyrin (54–94 aa residues; F) antibodies in the adult mouse cerebellar cortex. GL, Granular layer; ML, molecular layer; PC, Purkinje cell layer. Scale bars: 20 μm.

Figure 8.

Double immunofluorescence showing developmental switching of postsynaptic receptor phenotypes in PC somata. A₁–A₃, P7. B₁–B₃, P9. C₁–C₃, F, G₁, G₂, P12. D₁–D₃, P15. E₁–E₃, P20. Double immunofluorescence was applied to paraffin sections for GluA2 (red) and GABA_ARα1 (green) in A₁–E₃, GluA2 (red) and PSD-95 (green) in F, and gephyrin (red) and GABA_ARα1 (green) in G₁ and G₂. Arrows in A₁–E₃ indicate some GABA_ARα1 clusters lacking GluA2 labeling. Arrowheads in C₁ indicate fragmental GluA2 clusters, which are apposed closely to or piled up on GABA_ARα1 clusters. Arrows and arrowheads in G₁ and G₂ indicate somatic or dendritic clusters, respectively, that coexpress GABA_ARα1 and gephyrin. Asterisks indicate PC somata. Scale bars: 10 μm.

Figure 9.

Triple immunofluorescence examining the anatomical relationship of GluA2 and GABA_ARα1 clusters with CF or BF terminals at P12. A–C, Triple immunofluorescence for GluA2 (red), GABA_ARα1 (green), and VGluT1 (blue). D–F, Triple immunofluorescence for GluA2 (red), GABA_ARα1 (green), and VIAAT (blue). Orthogonal views of A and D are shown to the right and bottom. Boxed regions in A and D are enlarged in B₁–C₃ and E₁–F₃. Large receptor clusters in the apical portion of PC somata (red or green arrowheads) are shown in B and E, whereas fragmental receptor clusters in the basal portion (red or green arrows) are in C and F. Scale bars: 5 μm.

Figure 10.

Postembedding immunogold for AMPA receptor and PSD-95 in PC synapses at P12. A–C, Single-labeling immunogold for AMPA receptor (ϕ = 10 nm; A) or double-labeling immunogold for AMPA receptor (ϕ = 10 nm) and VIAAT (ϕ = 15 nm; B, C) at a CF–spine synapse on the soma (A), BF–spine synapse (B), and BF–soma synapse (C). D–F, Double-labeling immunogold for PSD-95 (ϕ = 10 nm) and VGluT2 (ϕ = 15 nm) or VIAAT (ϕ = 15 nm) at a CF–spine synapse on the soma (D), BF–spine synapse (E), and BF–soma synapse (F). Note the occasional labeling of PSD-95 at the asymmetrical contact of the BF–spine synapse (E). Edges of the PSD and immunogold particles for AMPA receptor and PSD-95 are indicated by arrowheads and arrows, respectively. G, H, Summary bar graphs comparing the labeling density for AMPA receptor (G) and PSD-95 (H) at CF–spine, BF–spine, BF–soma, and BF–dendrite synapses. Error bars indicate SEM. Numbers of immunogold-labeled synapses and total analyzed synapses are indicated in parentheses. The number of immunogold particles (mean ± SEM) for pan-AMPAR per synapse is 4.4 ± 1.0, 10.4 ± 1.9, 0.5 ± 0.3, 0.7 ± 0.2, and 0.1 ± 0.1 in CF–somatic spine, CF–dendritic spine, BF–spine, BF–soma, and BF–dendrite synapses, respectively. The number of immunogold particles for PSD-95 per synapse is 3.5 ± 1.3, 5.8 ± 1.3, 1.5 ± 0.4, 0.3 ± 0.2, and 0.1 ± 0.1 in CF–somatic spine, CF–dendritic spine, BF–spine, BF–soma, and BF–dendrite synapses, respectively. N.S., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (Mann–Whitney U test). Scale bars: 200 nm.

Figure 11.

Postembedding immunogold for GABA_ARα1 and gephyrin in PC synapses at P12. A–C, Double-labeling immunogold for GABA_ARα1 (ϕ = 10 nm) and VGluT2 (ϕ = 15 nm) or VIAAT (ϕ = 15 nm) at CF–spine (A), BF–spine (B), and BF–soma (C) synapses. Note the GABA_ARα1 labeling at the asymmetrical contact of the BF–spine synapse (C). D–G, Double-labeling immunogold for gephyrin (ϕ = 10 nm) and VIAAT (ϕ = 15 nm) or VGluT2 (ϕ = 15 nm) at CF–spine (D), BF–spine (E), BF–soma (F), and BF–dendrite (G) synapses. Note the GABA_ARα1 labeling at the asymmetrical contact of the BF–spine synapse (C). Edges of the PSD and immunogold particles for GABA_ARα1 and gephyrin are indicated by arrowheads and arrows, respectively. H, I, Summary bar graphs comparing the labeling density for GABA_ARα1 (H) at CF–spine, BF–spine, BF–soma, and BF–dendrite synapses. The number of immunogold particles (mean ± SEM) for GABA_ARα1 per synapse is 0.1 ± 0.0, 0.2 ± 0.1, 1.7 ± 0.5, 3.7 ± 0.4, and 6.0 ± 0.8 in CF–somatic spine, CF–dendritic spine, BF–spine, BF–soma, and BF–dendrite synapses, respectively. The number of immunogold particles for gephyrin per synapse is 0.6 ± 0.3, 0.3 ± 0.2, 1.4 ± 0.6, 2.4 ± 0.6, and 21.4 ± 3.6 in CF–somatic spine, CF–dendritic spine, BF–spine, BF–soma, and BF–dendrite synapses, respectively. N.S., p > 0.05; **p < 0.01; ***p < 0.001 (Mann–Whitney U test). Scale bars: 200 nm. PCS, Purkinje cell soma.

Figure 12.

Double-labeling postembedding immunogold for AMPA receptor and GABA_ARα1 in BF synapses at P12. A, B, Low- (A) and high-power (B) views of a BF terminal forming symmetrical contact with flat somatic surface and asymmetrical contact with somatic spine (asterisks). Consecutive images in B₁–B₃ demonstrate that clusters of immunogold particles for AMPA receptor (ϕ = 10 nm; arrowheads) are juxtaposed with GABA_ARα1 clusters (ϕ = 20 nm; arrows) on the PC soma under the same BF terminal. Scale bars: A, B₃ (for B₁–B₃), 500 nm. PCS, Purkinje cell soma.

Figure 13.

Summary illustrations on developmental switching of perisomatic PC synapses and receptor phenotypes. At P7 and P9, the predominance of CF–spine (CF–Sp) synapses corresponds to the pericellular nest stage of CF innervation. At P12, perisomatic CF–spine synapses are displaced to the apical somatic portion and shaft dendrites (the capuchon stage), whereas BF–spine synapses (BF–Sp) and BF–soma synapses (BF–Sm) increase reciprocally. At this stage, fragmental GluA2 and GABA_ARα1 clusters are juxtaposed preferentially at basal somatic portion. Juxtaposed receptor clusters are often covered with the same BF terminals. At P15, the major perisomatic synapses are BF synapses composed of BF–spine and BF–soma synapses, and most CF–spine synapses are eliminated from PC somata (the dendritic stage). At P20, most perisomatic synapses become BF–soma synapses, and the pinceau is established, thus attaining the adult type of innervation. Free spines surrounded by BFs or BGs also appear transiently during the period of CF-to-BF switchover, and may play some role in the restructuring of perisomatic innervation.