Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells in vitro and in vivo

Abstract

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

Figure 1.

Excitatory synapse formation induced by exogenous Cbln1 in cultured Purkinje cells. A, Representative images of cultured Purkinje cells at 21 DIV, prepared from wild-type (WT) and cbln1-null mice [knock-out (KO)] and immunostained against calbindin (red) and synaptophysin (green). The three columns on the right show enlarged views of the boxed regions in the two left columns. The far right column shows the images taken with reduced gain and saturation (less sat.) settings. B, The intensity of synaptophysin immunoreactivity is strong at the distal dendrites in WT cells but significantly weaker in cbln1-null cells. Incubation with exogenous Cbln1 (3 μg/ml) in the medium for 7 d completely restored the reduction in the synaptophysin signal. The data were normalized to the averages of the wild-type control cells for each experiment (n = 24 cells; 3 independent experiments; ***p < 0.0001). C, Images of distal dendrites of cultured Purkinje cells stained with calbindin (red) and synaptophysin (green). Incubation with a mutant Cbln1 (dS-Cbln1; 3 μg/ml) did not change synaptophysin signals in cbln1-null Purkinje cells. D, E, Images of distal dendrites of cultured Purkinje cells stained with calbindin (red) and either VGluT1 (D, green) or VGAT (E, green). The far right column shows images taken with reduced gain and saturation settings. Application of Cbln1 to the medium of cbln1-null cells increased the signals of VGluT1 (D) but not of VGAT (E). Scale bars: A, two far left columns, 30 μm (the entire images of the Purkinje cell); A, three columns on the right, C–E, 10 μm (the enlarged view of the distal dendrites).

Figure 2.

Continued presence of Cbln1 is necessary to maintain cbln1-null Purkinje cell synapses in vitro. A, Experimental design. Cbln1-null cerebellar cultures were incubated with exogenous HA-Cbln1 (3 μg/ml) for 7 d from 14 DIV (gray bars). The cells were fixed on days 1, 3, or 7 after the removal of HA-Cbln1 at 21 DIV (wash 1 d, wash 3 d, and wash 7 d). As a negative control, the cells were incubated with medium containing no HA-Cbln1 for 7 d (No treat) or for indicated durations (NC, negative control; unfilled bars). As a positive control, HA-Cbln1 was added (3 μg/ml) to the medium at various time points (+Cb; filled bars). The culture medium was replaced and rinsed with the appropriate medium at 21 DIV (the asterisk indicates wash). B, Representative images of distal dendrites of Purkinje cells stained with calbindin (red) and synaptophysin (green). The synaptophysin signal on the dendrites gradually decreased. Scale bars: top, 30 μm; bottom, 10 μm. C, Average intensity of synaptophysin signal at the distal dendrites of the Purkinje cells. The effect of removing HA-Cbln1 was estimated by comparing with the no-treatment control (No treat) or negative control (NC) for each group. One day after the removal of exogenous Cbln1, the signal was still comparable with the level before removal, whereas 7 d after the removal, the signal declined to the value of the negative control (n = 24 cells; 3 independent experiments; ***p < 0.001). n.s., No significant difference.

Figure 3.

Formation of functional PF-Purkinje cell synapses in acute slices of cbln1-null mice after incubation with exogenous Cbln1. A–C, mEPSCs from Purkinje cells were recorded in the slices from cbln1-null (KO) and wild-type (WT) mice (P9–P12) after incubation in aCSF containing 30 μg/ml HA-Cbln1 for 6–10 h. A, Sample traces of mEPSCs recorded from Purkinje cells using whole-cell recordings. B, The average frequency of the mEPSCs in cbln1-null cells was significantly increased by exogenous wild-type HA-Cbln1 (WT-Cbln1) and became comparable with that in the WT control. No change was observed with a mutant Cbln1 (dS-Cbln1; 30 μg/ml). **p < 0.01. C, The average amplitude of the mEPSCs was not changed by exogenous HA-Cbln1. D, Purkinje cells were whole-cell voltage clamped to record EPSCs evoked by the stimulation of PFs, the axon bundles of granule cells. Acute slices from P13 to P15 mice were treated as described above before recording. E, The amplitude of PF-EPSCs in cbln1-null cells increased to the wild-type level after incubation with exogenous HA-Cbln1. Insets show representative PF-EPSC traces recorded from Purkinje cells clamped at −70 mV in response to increasing stimulus intensities. ***p < 0.001. n.s., No significant difference. F, Summarized plots of the PPF ratio. The PPF ratio was defined as the amplitude of the second EPSC divided by that of the first EPSC at interpulse intervals of 50 ms. The PPF ratio, which was increased in cbln1-null cells, did not change with exogenous HA-Cbln1. The inset shows representative PF-EPSC traces in response to paired pulses. **p < 0.01. n.s., No significant difference.

Figure 4.

Anatomical recovery of PF-Purkinje cell synapses in acute slices of cbln1-null mice after the incubation with exogenous Cbln1. A, Electron micrographs of the molecular layer of the acute cerebellar slices prepared from cbln1-mice aged P14 after incubation with 30 μg/ml exogenous HA-Cbln1 for 8 h. In control slices, free spines (f) were observed frequently. Treatment with HA-Cbln1 significantly increased normal spines (n) and reduced the ratio of free spines. Scale bar, 500 nm. B, Density of normal, free, and mismatched spines in cbln1-null acute cerebellar slices after incubation with HA-Cbln1 or control medium for 8 h. n = 10–11 sections for each treatment (n = 1 mouse). ***p < 0.001.

Figure 5.

Improvement of cerebellar ataxia in adult cbln1-null mice after the subarachnoidal injection of Cbln1. A, Distribution of injected Cbln1, stained with anti-HA antibody (red), in the cerebellum at 24 h after injection. Purkinje cells were stained with calbindin (green). The injected Cbln1 was localized in all the cerebellar lobules. Enlarged images show the punctate signal of HA-Cbln1 localized on the dendrites of Purkinje cells. Scale bars: two far left columns, 1 mm; third column from the left, 30 μm; fourth column, 10 μm. B, Injected Cbln1 from the P2 fraction of the whole cerebellum was detected using immunoblotting and an antibody against HA (a) or Cbln1 (b) at 1, 2, 4, and 7 d after the injection. The signal in a was normalized with that of actin and further normalized to the value at 1 d after the injection (c). The injected HA-Cbln1 level decreased to <10% on day 7 (n = 3 mice). C, Representative gait pattern of a cbln1-null mouse before and 2 d after the injection. The hindpaws were marked with black paint. The irregular, shortened gait skips were markedly improved after the injection of Cbln1. L, Left paw; R, right paw. Numbers indicate step counts. D, Results on the rotarod test after the injection of Cbln1 medium (KO+Cbln1) or the control medium (KO+control). The average time on the rotarod at 20 rpm was calculated from four trials (n = 4 mice).

Figure 6.

Complete but transient structural restoration of PF-Purkinje cell synapses in cbln1-null mice by subarachnoidal injection of Cbln1. A, Electron micrographs of the molecular layer in cbln1-null mice aged P42–P54 at 2 d after the injection of either control or HA-Cbln1 and at 35 d after the injection of HA-Cbln1. In control mice, free spines (f) and mismatched spines (m) were found frequently. Two days after the injection of HA-Cbln1, the number of normal spines (n) had increased, and PF terminals that were in contact with multiple spines (*) were observed frequently. Thirty-five days after the injection, the number of normal spines had decreased to the original level. B, Percentage of normal and free spines in cbln1-null mice at 1, 2, 4, 7, 12, and 35 d after the injection of HA-Cbln1 (n = 3 mice). The effect of Cbln1 was compared with that in age-matched cbln1-null mice that did not receive treatment (no treat.). *p < 0.05; ^# p < 0.001; ^## p < 0.0001. C, Percentage of mismatched spines estimated using serial section electron microscopy in cbln1-null mice without treatment (n = 3 mice) and 2 d after the injection of either the control or Cbln1 medium (n = 1 mouse). The number of mismatched spines decreased with the injection of HA-Cbln1.

Figure 7.

Functional restoration of PF-Purkinje cell synapses in cbln1-null mice by the injection of Cbln1. Two days after the injection, PF-EPSCs were recorded from Purkinje cells using the whole-cell patch-clamp technique. The input–output relationship of PF-EPSCs in cbln1-null cells became similar to those in wild-type cells after a single injection of HA-Cbln1. The injection of control medium had no effect. ***p < 0.001.