Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum

Abstract

Carbonic anhydrase related protein 8 (Car8) is known to be abundantly expressed in Purkinje cells (PCs), and its genetic mutation causes a motor coordination defect. To determine the underlying mechanism, we analyzed the mouse cerebellum carrying a Car8 mutation. Electrophysiological analysis showed that spontaneous excitatory transmission was largely diminished while paired pulse ratio at parallel fiber-PC synapses was comparable to wild-type, suggesting functional synapses have normal release probability but the number of functional synapses may be lower in mutants. Light microscopic study revealed an abnormal extension of climbing fibers to the distal PC dendrites. At the ultrastructural level, we found numerous PC spines not forming synapses primarily in distal dendrites and occasionally multiple spines contacting a single varicosity. These abnormalities of parallel fiber-PC synapses may underlie the functional defect in excitatory transmission. Thus, Car8 plays a critical role in synaptogenesis and/or maintenance of proper synaptic morphology and function in the cerebellum.

Fig. 1

Immunohistochemical staining for Purkinje cell (PC) specific markers in the cerebellum of rig mice. (A, B) Wild-type (A) and rig (B) cerebellum stained with anti IP3 receptor (IP3R, green). Insets show higher magnification view of PCs. (C, D) The lobule IX of wild-type (C) and rig (D) cerebellum stained with anti Calbindin-D (green). Cell nuclei are counterstained with DAPI (blue) in A–D. (E, F) Calbindin-D staining showing the morphology of PC dendritic spines in wild-type (E) and rig (F) mice. Insets show higher magnification view of individual spines. Scale bars: 5 μm.

Fig. 2. Excitatory transmission is diminished in Car8^wdl mutants

(A1) Samples of mEPSCs recorded from Purkinje cells in wild-type or Car8^wdl mutant mice as indicated. (A2, A3) Cumulative plots of inter-event intervals (A2) and the amplitude (A3) of mEPSCs. The frequency of mEPSCs is significantly less in Car8^wdl mutants (A2) whereas there is no difference in the amplitude (A3). (B1) Representative recordings of EPSCs evoked by paired pulses. Both WT and Car8^wdl animals show paired pulse facilitation. (B2) Summary graph of paired pulse ratio, demonstrating no statistical difference between the two groups. Error bars in A2, A3 and B2 represent standard error.

Fig. 3

CF and PF labeling in the wild-type (A, C, E) and Car8^wdl (B, D, F) cerebellum. (A, B) Double immunofluorescence for VGLUT2 (green) and Calbindin (red). Note that the distribution of VGLUT2-labeled CF terminals is expanded distally in the molecular layer of Car8^wdl (B) compared to wild-type (A) mice. (C, D) Higher magnification view of VGLUT2 (green) and Calbindin (red) labeling. While VGLUT2 is mainly localized along the thick, proximal dendrites in wild-type mice (C), VGLUT2-labeled terminals are also associated with thinner, distal dendrites in Car8^wdl mice (D, arrowheads). Note an increase of VGLUT2-labeled puncta in the Car8^wdl molecular layer. (E, F) Double imunofluorescence for VGLUT1 (green) and Calbindin (red). No overt difference in the distribution of VGLUT1-labeled PF terminals was detected between wild-type (E) and Car8^wdl (F) mice.

Fig. 4

Electron micrographs of free dendritic spines in Car8^wdl cerebellum. (A–C) Numerous free spines are observed in clusters at various positions in the molecular layer of the cerebellum. Representative images are shown from distal (A) and proximal (B) dendritic field to the PC soma. Asterisks denote free spines. Note that free spines contain postsynaptic density (arrowheads) and endoplasmic reticulum (arrows). Scale bars: 1μm. (C) Number of PC dendritic spines per area in wild type (WT) and Car8^wdl mice. (D) Distribution of free spines in the molecular layer of Car8^wdl mice. Numbers of free spines per area in the region adjacent to the distal border of the molecular layer (distal) and the region adjacent to the PC soma (peri-PC soma) are shown. Error bars represent standard deviation. (E–L) Serial electron micrographs of PC dendritic spine unattached to any nerve terminals (free spine) in Car8^wdl mice. Scale bar: 500nm.

Fig. 5

Electron microscopic analysis of excitatory synaptic contacts in wild-type and Car8^wdl cerebellum. In wild-type mice, parallel fiber varicosities interact with one (A) or two (B) dendritic spines of the PCs. In the Car8^wdl cerebellum, in addition to the varicosities interacting with one (C) or two (D) dendritic spines, those contacting more than two spines (E, F) are observed. S: dendritic spines, PFV: parallel fiber varicosity. Scale bar: 500 nm. As summarized in (G), the percentage of multiple synaptic varicosities (2–4 dendritic spines contacting 1 varicosity) against total synaptic interactions is significantly higher (**, p<0.005) in Car8^wdl mice. (H) shows the classification of multiple synaptic varicosities into 1:2 (presynapse vs postsynapse), 1:3 and 1:4, and the percentage of multiple synaptic varicosities belonging to each class. ** and * denote statistical significance with p<0.005 and p<0.05, respectively.