Remodeling of dendrites and spines in the C1q knockout model of genetic epilepsy

Abstract

Purpose: To determine whether developmental synaptic pruning defects in epileptic C1q-knockout (KO) mice are accompanied by postsynaptic abnormalities in dendrites and/or spines.

Methods: Immunofluorescence staining was performed on biocytin-filled layer Vb pyramidal neurons in sensorimotor cortex. Basal dendritic arbors and their spines were reconstructed with NEUROLUCIDA software, and their morphologic characteristics were quantitated in Neuroexplorer.

Key findings: Seven to nine completely filled pyramidal neurons were analyzed from the wild-type (WT) and C1q KO groups. Compared to WT controls, KO mice showed significant structural modifications in their basal dendrites including (1) higher density of dendritic spines (0.60 ± 0.03/μm vs. 0.49 ± 0.03/μm dendritic length in WT, p < 0.05); (2) remarkably increased occurrence of thin spines (0.26 ± 0.02/μm vs. 0.14 ± 0.02/μm dendritic length in control, p < 0.01); (3) longer dendritic length (2,680 ± 159 μm vs. 2,119 ± 108 μm in control); and (4) increased branching (22.6 ± 1.9 vs. 16.2 ± 1.3 in WT at 80 μm from soma center, p < 0.05; 12.4 ± 1.4 vs. 8.2 ± 0.6 in WT at 120 μm from soma center, respectively, p < 0.05). Dual immunolabeling demonstrated the expression of putative glutamate receptor 2 (GluR2) on some thin spines. These dendritic alterations are likely postsynaptic structural consequences of failure of synaptic pruning in the C1q KO mice.

Significance: Failure to prune excessive excitatory synapses in C1q KO mice is a likely mechanism underlying abnormalities in postsynaptic dendrites, including increased branching and alterations in spine type and density. It is also possible that seizure activity contributes to these abnormalities. These structural abnormalities, together with increased numbers of excitatory synapses, likely contribute to epileptogenesis in C1q KO mice.

Keywords: C1q; Dendrite; Genetic epilepsy; Pruning; Spine.

Figure 1. Increased dendritic length in C1q KO vs. WT mice

A: Confocal projection images of dendritic arbors of biocytin (green) – filled Pyr cells from WT (left) and KO (right) mice. B: Neuroexplorer projection images of 3-D reconstructed basal dendrites shown in A. C: Bar graph of the dendritic length of KO (n=8) and control (n=9) Pyr cells. *: p<0.05.

Figure 2. Increased dendritic branching in KO vs. WT mice

A: Concentric circle lines (red) overlapping 3-D reconstructed basal dendrites. The increment between Sholl lines was 40 µm. B: Bar graph of the numbers of intersections between dendrites and Sholl lines at three distances from the soma center: 40, 80, and 120 µm.

Figure 3. Increased density of total dendritic spines and thin spines in KO vs. WT mice

A: Three types of spines: thin (left), mushroom (middle), and stubby (right). Arrows indicate spine heads. B: Confocal images of dendritic segments from a WT (B1) and a KO (B2) Pyr cells. C: Reconstructed images of three distinct types of spines along corresponding dendritic segments shown in B (thin: red, mushroom: blue, stubby: green). For visual clarity, stubby spines are indicated by green arrows. D: Bar graph of the density of total spines (D1) and individual types of spines (D2) from 7 KO and 8 WT Pyr cells. **: p<0.01.

Figure 4. Localization of glutamate receptor 2 (GluR2) in dendritic thin spines in a biocytin-filled C1q KO Pyr cell

A: Confocal image of a dendritic segment with dual immunolabeling for biocytin (green) and GluR2 (red), representative of findings in 6 biocytin-fillled Pyr cells. Three representative sites of colocalization on thin spines (1, 2 and 3) were enlarged on right and indicated by arrows. Colocalization of the two immunoreactivities (yellow) is present on spines indicating that they express GluR2. Calibration = 1 µm for A and 0.5 µm for images 1–3.