Combined DiI and Antibody Labeling Reveals Complex Dysgenesis of Hippocampal Dendritic Spines in a Mouse Model of Fragile X Syndrome

Abstract

Structural, functional, and molecular alterations in excitatory spines are a common hallmark of many neurodevelopmental disorders including intellectual disability and autism. Here, we describe an optimized methodology, based on combined use of DiI and immunofluorescence, for rapid and sensitive characterization of the structure and composition of spines in native brain tissue. We successfully demonstrate the applicability of this approach by examining the properties of hippocampal spines in juvenile Fmr1 KO mice, a mouse model of Fragile X Syndrome. We find that mutant mice display pervasive dysgenesis of spines evidenced by an overabundance of both abnormally elongated thin spines and cup-shaped spines, in combination with reduced density of mushroom spines. We further find that mushroom spines expressing the actin-binding protein Synaptopodin-a marker for spine apparatus-are more prevalent in mutant mice. Previous work identified spines with Synaptopodin/spine apparatus as the locus of mGluR-LTD, which is abnormally elevated in Fmr1 KO mice. Altogether, our data suggest this enhancement may be linked to the preponderance of this subset of spines in the mutant. Overall, these findings demonstrate the sensitivity and versatility of the optimized methodology by uncovering a novel facet of spine dysgenesis in Fmr1 KO mice.

Keywords: DiIC18; Fmr1 knockout mouse; Fragile X Syndrome; dendritic spines; excitatory synapses; hippocampus; synaptopodin.

Figure 1

Workflow of DiIC₁₈ staining combined with fluorescent immunolabeling. (a) Overview of the 1-step protocol for visualization and morphometric analysis of dendritic spines in rodent brain tissue. Shown in the middle panel is a representative confocal image of an area of the hippocampus from a coronal section of WT mouse brain stained with DiIC₁₈; scale bar, 5 µm. The bottom panel is a graphical representation of different types of dendritic spines. (b) Overview of a 2-step protocol for morphometric analysis combined with detection of synaptic proteins. Shown in the middle panel is a representative confocal image of an area of the hippocampus from a coronal section stained with DiIC₁₈ and immunolabeled with anti- Synpo antibody: scale bar 5 µm, magnified inset 2 µm. Mushroom (M) spines M−, Synpo-negative mushroom spine; M+ Synpo-positive mushroom spine. Illustrations in the top panels of (a,b) were created with Biorender.com (accessed on 25 August 2022).

Figure 2

Visualization of Synpo-containing spines and VGluT2- or Synpr-positive terminals at hippocampal synapses ex vivo. (a) Representative confocal images of WT hippocampal dendrites stained with DiIC₁₈ and labeled with anti-Synpo together with either anti-VGluT2 (left panels) or anti-Synpr (right panels). Shown are individual channels (DiIC₁₈ in blue) and the overlay of the three channels (Merge); boxed areas are shown magnified in (b), scale bars 5 µm. (b) Magnified images of boxed areas in (a); scale bars, 2 µm. Arrows point to regions of Synpo/VGluT2 or Synpo/Synpr overlap.

Figure 3

DiIC₁₈ staining reveals dysgenesis of hippocampal spines in juvenile Fmr1 KO mice. (a) Representative confocal image of a dendritic segment from WT mouse hippocampus, stained with DiIC₁₈; scale bar, 2 µm. Labels indicate the different types of dendritic protrusions identified: M mushroom spines, T thin spines, S stubby spines, and F filopodia. (b) Quantification of total dendritic protrusions per dendritic length (µm) in WT and Fmr1 KO mice. Differences were evaluated by Student’s t-test; ** p = 0.006, N = 3 mice per group. (c) Quantification of spines density per dendritic segment and categorization by morphology as thin, mushroom, stubby spines, and filopodia. Differences were evaluated by two-way ANOVA followed by Tukey’s post-hoc multiple comparisons test * p < 0.05 KO vs. WT, °°°° p < 0.0001 WT vs. WT, #### p < 0.0001 KO vs. KO. The effect of genotype (p = 0.0041) and spine’s type (p < 0.0001) is significant. The interaction between the main factors is significant (p < 0.0001); N = 3 mice per group. (d) Pie charts summarizing the relative proportion (%) of the four most common types of spines analyzed in (c).

Figure 4

Morphological alterations of thin and mushroom spines in the Fmr1 KO mouse hippocampus. (a) Representative confocal images of dendritic branches stained with DiIC₁₈ from WT and Fmr1 KO hippocampi; scale bars, 5 µm. Boxed regions are displayed in magnified insets below; scale bars, 2 µm. Arrows indicate the length and head width measurements; d, diameter of spine heads. (b) Quantification of the length of thin spines in WT and Fmr1 KO littermates. (c) Quantification of mushroom spines head width (diameter) in WT and Fmr1 KO mice. Differences were evaluated by Student’s t-test * p = 0.018, *** p < 0.0008, N = 3 mice per group.

Figure 5

Abnormal prevalence of branched spines in the hippocampus of juvenile Fmr1 KO mice. (a) Representative confocal images of dendritic branches stained with DiIC₁₈ from WT and Fmr1 KO hippocampi; scale bars, 5 µm. Boxed regions are displayed in magnified insets (right panels), scale bars, 2 µm. Arrows point to branched, cup-shaped spines. (b) Quantification of the relative density of branched (cup-shaped) spines per neurite length (µm) in WT and Fmr1 KO mice. Differences were evaluated by Student’s t-test ** p = 0.0043, N = 3 mice per group. (c) Pie charts summarizing the relative proportion (%) of dendrites with branched spines relative to all dendritic branches examined in (b).

Figure 6

Increased abundance of mushroom spines containing Synpo in juvenile Fmr1 KO mice. (a) Representative confocal images of dendritic branches stained with DiIC₁₈ and immunolabeled with anti-Synpo from WT and Fmr1 KO hippocampi; scale bars, 5 µm. (b) Magnified images of boxed areas in (a) i, scale bars 2 µm. Arrows point to Synpo-positive (S+) mushroom spines. (c) Quantification of Synpo-positive (S+) mushroom spines relative to the total number of mushroom spines in WT and Fmr1 KO mice. Differences were evaluated by Student’s t-test * p = 0.038, from N = 3 mice per group.