Impaired parallel fiber-->Purkinje cell synapse stabilization during cerebellar development of mutant mice lacking the glutamate receptor delta2 subunit

Abstract

The glutamate receptor delta2 subunit (GluRdelta2) is specifically expressed in cerebellar Purkinje cells (PCs) from early developmental stages and is selectively localized at dendritic spines forming synapses with parallel fibers (PFs). Targeted disruption of the GluRdelta2 gene leads to a significant reduction of PF-->PC synapses. To address its role in the synaptogenesis, the morphology and electrophysiology of PF-->PC synapses were comparatively examined in developing GluRdelta2 mutant and wild-type cerebella. PCs in GluRdelta2 mutant mice were normally produced, migrated, and formed spines, as did those in wild-type mice. At the end of the first postnatal week, 74-78% of PC spines in both mice formed immature synapses, which were characterized by small synaptic contact, few synaptic vesicles, and incomplete surrounding by astroglial processes, eliciting little electrophysiological response. During the second and third postnatal weeks when spines and terminals are actively generated, the percentage of PC spines forming synapses attained 98-99% in wild type but remained as low as 55-60% in mutants, and the rest were unattached to any nerve terminals. As a result, the number of PF synapses per single-mutant PCs was reduced to nearly a half-level of wild-type PCs. Parallelly, PF stimulation less effectively elicited EPSCs in mutant PCs than in wild-type PCs during and after the second postnatal week. These results suggest that the GluRdelta2 is involved in the stabilization and strengthening of synaptic connectivity between PFs and PCs, leading to the association of all PC spines with PF terminals to form functionally mature synapses.

Fig. 1.

Cerebellar histology and Purkinje cell cytology in the GluRδ2 mutant (A, C) and wild-type mouse (B, D). A, B, Nissl-stained sagittal cerebellar sections. Note reductions in thickness of the granular and molecular layers in the mutant cerebellum. Rostral is to the right, and dorsal is at the top. C, D, Confocal laser scanning microscopic images of PCs immunostained for spot 35/calbindin. Note elaborate branching of PC dendrites studded with numerous spines. Mo, Molecular layer. Scale bars:B, 0.5 mm; D, 20 μm.

Fig. 2.

Serial electron micrographs of the molecular layer in the GluRδ2 mutant mouse at P35. A, Image from a set of serial sections. In the mutant mouse, spines (s) protruding from PC dendrites (d) occupy the molecular layer as densely as in the wild-type mouse (Fig. 3A). Note some profiles of unattached spines possess PSD-like dense materials (arrowheads) under the cell membrane.B–I, PC spine (asterisks) unattached to any nerve terminals. Note that small PSD-like dense material is seen under the postsynaptic membrane (arrowheads).J–Q, PC spine (asterisks) in contact with PF terminal (pf). Scale bars, 0.5 μm.

Fig. 3.

Serial electron micrographs of the molecular layer in a wild-type mouse at P35. A, Image from a set of serial sections. B–I, PC spine (asterisks) in contact with PF terminal (pf). s, PC spine. Scale bars, 0.5 μm.

Fig. 4.

Parallel fiber→Purkinje cell synapses in the wild-type mouse at P35. Asterisks indicate PC spines in contact with PF terminals. PF→PC synapses between one terminal and two spines are occasionally found in wild-type mice. Scale bar, 0.5 μm.

Fig. 7.

Postnatal changes in the contact ratio between the PF terminal and PC spine. A, P7; B, P14;C, P21; D, P35. The total number of PF→PC synapses analyzed is shown in the top right corner. Statistics, χ² test.

Fig. 5.

Electron micrographs showing developing PF→PC synapses in the GluRδ2 mutant (A, C) and wild-type (B, D) mice. A, B, P7. Note small synaptic contacts (arrowheads), which are not fully surrounded by astroglial investments in both mice. C,D, P14. PF→PC synapses (arrowheads) develop normal structure in both mice. However, profiles of unattached PC spines (asterisks) are more obvious in the mutant mouse. Note that unattached spines often possess a small PSD-like condensation in the mutant mouse but not in the wild-type mouse. Scale bar, 0.5 μm.

Fig. 6.

Postnatal changes in the percentage of contacted PC spines. The total numbers of PC spines analyzed from mutant and wild-type mice are n = 60 or 60 at P7,n = 122 or 116 at P14, n = 60 or 90 at P21, and n = 271 or 253 at P35, respectively. p = 0.32 at P7, 0.02 at P14 and P21, and 0.009 at P35. Statistics, Student’s t test.

Fig. 8.

Reduced electrophysiological responses to PF stimulation in GluRδ2 mutant PCs. Amplitudes of PF-EPSCs are plotted as a function of stimulus intensity in the wild-type (open circles) and GluRδ2 mutant (filled circles) PCs sampled from mice at P14–P17 (A;n = 10–12 for the wild-type, andn = 18–24 for the GluRδ2 mutant mice), P21–P22 (B; n = 18–20 for the wild-type, and n = 9–13 for the GluRδ2 mutant mice), and P30–P33 (C; n = 20–25 for the wild-type, and n = 19–27 for the GluRδ2 mutant mice). Each point represents the mean ± SEM.Asterisks indicate significant differences between the wild-type and GluRδ2 mutant mice (*p < 0.05; **p < 0.01, t test).Insets, Representative traces of PF-EPSCs with increasing stimulus intensities of 2, 6, 10, and 15 μA.