Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells

Abstract

The receptor tyrosine kinase TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), are critically important for growth, survival and activity-dependent synaptic strengthening in the central nervous system. These TrkB-mediated actions occur in a highly cell-type specific manner. Here we report that cerebellar Purkinje cells, which are richly endowed with TrkB receptors, develop a normal morphology in trkB-deficient mice. Thus, in contrast to other types of neurons, Purkinje cells do not need TrkB for dendritic growth and spine formation. Instead, we find a moderate delay in the maturation of GABAergic synapses and, more importantly, an abnormal multiple climbing fiber innervation in Purkinje cells in trkB-deficient mice. Thus, our results demonstrate an involvement of TrkB receptors in synapse elimination and reveal a new role for receptor tyrosine kinases in the brain.

Fig. 1

Normal layer formation in cerebella of trkB^-/- mice. (a) Genotyping of experimental animals by PCR. (b) Immunohistochemistry of Trk receptors and calbindin in cerebella from P7 trkB^+/+ and trkB^-/- mice. Trk receptors were stained using a rabbit pan-Trk antibody and calbindin using a monoclonal calbindin antibody. The pan-Trk antibody demonstrated the low abundance of other Trk receptors in trkB^-/- mice. Scale bar = 50 μm.

Fig. 2

Normal PC morphology in trkB^-/- mice. (a) Two-photon images of PCs filled with Alexa Fluor-488 in acute slices. P10 PCs have a small apical dendritic tree and several perisomatic dendrites (left). At P14, PCs display extensive branching in both trkB^+/+ (middle) and ^-/- mice (right). No perisomatic dendrites can be observed anymore. (b) Digitized reconstructions of PCs from the images in (a) that were used for the quantitative analysis of the PC morphology. The colors reflect the centrifugal branch order analysis, each order having its own color. (c) Bar graphs depicting mean values of morphometric parameters resulting from the analysis of the reconstructed PCs. From left to right: number of stem dendrites originating at the soma, apical length, total dendritic length, somatodendritic area, number of bifurcations, tree asymmetry index. (d) Circles with increasing diameter were placed on the dendritic trees starting in the center of the soma (top). The number of intersections with dendritic elements was calculated for each circle (bottom). Shown are the mean values per circle. The error bars depict the SEM per 10 μm. The Sholl distribution of the P14 trkB^+/+ PCs reflects the larger dendritic size of these cells compared to the P10 trkB^+/+ PCs and also the P14 trkB^-/- PCs (two-way ANOVA). (e) Cluster analysis using 17 cell morphological parameters according to Ward's method revealed that the P10 trkB^+/+ PCs formed their own cluster (light blue). The P14 trkB ^+/+ (dark blue) and ^-/- (red) PCs did not segregate into separate clusters. Unless otherwise stated, the mean ± s.d. is shown. Significant difference with the P10 trkB^+/+ PCs is depicted by * and between P14 trkB^+/+ and trkB^-/- PCs by #. Unless otherwise stated, we used ANOVA with Tukey's post-test. The threshold for significance was set at 0.05. For this analysis, 10 P10 trkB^+/+ PCs, 13 P14 trkB^-/- and 7 trkB^-/- PCs were used.

Fig. 3

Normal spine densities in trkB^-/- mice. Spiny branchlets are covered with spines that are mainly innervated by PFs. The density of the PF spines is similar in P14 trkB^+/+ (a) and ^-/- PCs. in trkB^+/+ PCs (b). Top: z-projections of three or four planes. Bottom: surface renderings of the complete z-stacks.

Fig. 4

Delayed maturation of GABAergic synapses in trkB ^-/- mice. (a) Recordings of mIPSCs in PCs in cerebellar slices from P7 trkB^+/+ and ^-/- mice in the presence of tetrodotoxin (500 nM), CNQX (20 μM) and APV (25 μM). (b) Superimposed example mIPSCs at a more expanded time scale. (c) Cumulative histograms, constructed from 75 mIPSCs per experiment, depicting the 10-90% rise time, the amplitude, the ^τdecay and the interval of the mIPSCs. All distributions were significantly different (two-way ANOVA). The mIPSCs in the trkB^-/- PCs were smaller and decayed slower, while they occurred less frequently than the trkB^+/+ mIPSCs. From both genotypes, seven PCs were used for the recordings of mIPSCs.

Fig. 5

Synaptic responses to parallel and climbing fiber inputs in P14 trkB^-/- mice. (a) Paired-pulse facilitation of PF-EPSCs (left) an paired-pulse depression of CF-EPSCs (right)in trkB^-/- mice. PFs were electrically stimulated in the molecular layer and CFs in the granular layer. (b) CF activation elicits a complex spike in PCs from trkB^-/- mice. This complex spike is accompanied by a rise in [Ca²⁺]_i. The soma was kept out of focus to reduce phototoxic damage. Bar = 100 μm. (c) The CF-EPSC is mediated exclusively by AMPA receptors, since it could be blocked by the addition of CNQX (20 μM). After washing out CNQX for ~10 minutes, the CF-EPSC partially recovered.

Fig. 6

Abnormal multiple CF innervation in trkB^-/- mice at P14. In P14 trkB^+/+ mice, most PCs are innervated by a single, large CF. (a) Representative recording from a P10 trkB^+/+ PC, showing no response to a weak stimulus (left), a moderately large response to an intermediate stimulus (middle) and a large response to a strong stimulus (right). Analysis of a large range of stimulation intensities reveals that this PC receives input from two individual CFs (bottom). (b) Representative recording from a P14 trkB^+/+ PC, receiving one CF input and of a P14 trkB^-/- PC receiving two individual CF inputs (c). (d) At P10, the large majority of trkB^+/+ PCs is innervated by more than 1 CF. In contrast, at P14, most trkB^+/+ PCs are already mono-innervated. The phenotype of P14 trkB^-/- PCs is very similar to that of P10 trkB^+/+ PCs, demonstrating a clear reduction in the rate of developmental CF synapse elimination.