Dendritic spine density is altered in a mouse model of Cockayne syndrome

Cockayne syndrome (CS) (MIM 133540, 216400) is an autosomal recessive syndrome that features growth failure, neurological dysfunction, premature ageing and dementia [1]. Many CS cases are associated with mutations in the gene that encodes for Cockayne syndrome B (CSB), which functions in DNA repair [2]. Mice deficient in CSB exhibit progressive loss of Purkinje cells, p53 positivity in Purkinje cells and dysmyelination in the brain [3,4]. In our pilot study, we further characterize neurodegeneration in CSB-deficient mice by examining their dendritic spines. In another mouse model of deficient DNA repair – excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1)-deficient mice, there is impaired neuronal plasticity [5]. Hypothesizing that the CSB DNA repair pathway similarly influences neuronal structures involved in plasticity, we perform a morphometric analysis of dendritic spines of Golgistained neurones in Csb-/(knockout, KO) mice. Animal experimentation was performed in accordance with Canadian Council on Animal Care regulations. Experimental procedures were approved by the University of Toronto Animal Care Committee. Three KO and three Csb+/+ (wild-type, WT) mice were analysed. KO mice were bred, genotyped from tail snips and sacrificed at 6 months of age, as previously described [4,6]. Brain sections were analysed by Golgi staining: each mouse brain was placed into osmium dichromate working solution (20 ml 2% osmium tetroxide, 100 ml 3.2% potassium dichromate) for 7 days immediately following dissection. Tissue was incubated in 0.75% silver nitrate for 24 h, dehydrated in 70% ethanol for 2 h, embedded in paraffin blocks, sectioned sagittally at 25–30 um thickness with a sliding microtome (Leica Microsystems, Buffalo Grove, IL, USA) and mounted on slides. Slides were processed with 2¥ xylene solution, mounted with distyrene plasticizer xylene medium (EMS, Hatfield, PA, USA) and air-dried at room temperature for 2–3 weeks before quantitative analysis. Apical and basal dendrites of cortical neurones from frontal and parietal cortical regions were analysed with stereology-based software (Microbrightfield, Williston, VT, USA), coupled to a Zeiss Axioplan 2 image microscope (Carl Zeiss Microscopy, Germany) with motorized X, Y and Z-focus for high-resolution image acquisition and digital quantification. The experimenter performing the analyses was blinded to whether the specimen was KO or WT. The sampling process for candidate pyramidal cells and dendritic segments was conducted as follows: (i) a preview of the lateral-to-medial axis of the two regions of interest was performed under low and high magnification Zeiss objectives (10¥ and 20¥); (ii) cells with the least truncated distal dendrites were located under higher magnification Zeiss objectives (40¥ and 63¥); and (iii) dendritic tracing was conducted using a Zeiss 100¥ objective with immersion oil (numerical aperture = 1.25) followed by rigorous counting of spines across selected dendrite segments. In all cases, the soma was first identified as the origin of the branch order, and then each of the branch points of dendritic trees was traced. All spines protracting up to 300 um away from the nodes were counted. Next, the digital profile of neurone morphology underwent computer-based quantitative analysis (Microbrightfield), including dendrograms and spine counts to obtain spine density (number of spines per mm of dendrite segment) and affiliated dendritic branch orders. Two-tailed Student’s T-test was performed using GraphPad Prism (Version 5, GraphPad Software Inc., La Jolla, CA, USA) with P-value of 0.05. In the frontal and parietal cortices, dendritic spine densities of both basal and apical dendrites were reduced in KO compared with WT mice. Frontal cortex basal spine density was reduced from 0.75 0.02/mm in WT to 0.57 0.03/mm in KO mice (P < 0.001) (Figure 1A). Frontal cortex apical spine density was reduced from 0.81 0.04/mm in WT to 0.55 0.03/mm in KO mice (P < 0.001) (Figure 1A). Parietal cortex basal spine density was reduced from 0.89 0.04/mm in WT to 0.58 0.03/mm in KO mice (P < 0.001) (Figure 1B). Parietal cortex apical spine density was reduced from 0.95 0.04/mm in WT to 0.54 0.05/mm in KO mice (P < 0.001) (Figure 1B). There was no significant difference in spine density between frontal and parietal neurones in KO mice. Figure 1C illustrates the morphology of Golgi-stained basal and apical cortical neurones in WT and KO mice.