Ectopic migration of select populations of immature neurons in <i>Disc1</i>^100P/100P mice.

<p>(<b>A–C</b>) An analysis of doublecortin (DCX) positive cells prematurely migrating away from the subgranular zone (SGZ) (<b>A</b>, upper boundary of GCL demarcated with dashed line) reveal no significant (<b>B</b>) differences (ANOVA, p = 0.35, n = 7 wild type, n = 5 <i>Disc1</i>^31L/31L, n = 5 <i>Disc1</i>^100P/100P) between genotypes. Arrowheads point to DCX⁺ cell somas positioned in the GCL within 70 µm of the SGZ, while the arrow in <i>Disc1</i>^100P/100P section shows DCX⁺ cell positioned greater than 70 µm away from SGZ. (<b>C</b>) Binning of DCX⁺ cells in the GCL into 10 µm distance segments from 20 µm away from the SGZ reveal a small but significant (ANOVA F(2,14) = 5.180, p = 0.021; post-hoc Tukey p<0.05; n = 7 wild type, n = 5 <i>Disc1</i>^31L/31L, n = 5 <i>Disc1</i>^100P/100P) percentage of cells that migrate a distance greater than 70 µm in the <i>Disc1</i>^100P/100P mice compared to wild-type and <i>Disc1</i>^31L/31L mice. Data presented as mean ± SEM. Scale Bar 50 µm</p

Hippocampal Neuroanatomical Measurements between <i>Disc1</i>^31L/31L, <i>Disc1</i>^100P/100P and wild-type mice.

<p>A significant difference only in dentate granule cell layer thickness between wild-type and <i>Disc1</i>^100P/100P mice was noted (ANOVA F(2,20) = 4.07, P = 0.032; <i>post-hoc</i> Bonferroni p<0.05), though Student's <i>t</i> test indicated a strong correlation (p = 0.06) between wild-type and <i>Disc1</i>^31L/31L mice. Within the hippocampus, the thinning of the granule cell layer in the mutants is specific to the dentate gyrus, as no significant differences are observed between genotypes in the thickness of the pyramidal cell layer in the CA1 and CA3 regions, and the overall height and width of the hippocampus. Data presented as mean ± SEM.</p><p>Hippocampal Neuroanatomical Measurements between <i>Disc1</i>^31L/31L, <i>Disc1</i>^100P/100P and wild-type mice.</p

Hypothetical model for how DISC1 mutation may affect interacting proteins within the cell populations residing in the dentate gyrus.

<p>Neural stem cells (NSCs) generate neurons which extend processes across the granule cell layer through the generation of Tbr2⁺ intermediate progenitors (IPCs) as shown in the ‘wild type’ illustration. Our data suggests that the homozygous Q31L mutation in DISC1 leads to a loss of IPCs which may arise from a reduction in the formation of DISC1-GSK3β complexes in the NSCs leading to increased quiescence. With the homozygous L100P mutation, however, we note a normal number of IPCs, but instead observe alterations in the morphology and migration of the DCX⁺ postmitotic neurons, which we hypothesize may be due to changes in the NDEL1-LIS1 complex mediated by DISC1 interactions with PDE4.</p

Deficits in cell proliferation are restricted to <i>Disc1</i>^31L/31L mice.

<p>(<b>A</b>) Missense mutations do not affect expression of full length DISC1 in tissue lysates taken from the cortex and hippocampus, as shown using a C-terminal DISC1 antibody that recognizes a specific 100 kDa band in the adult mouse tissue brain homogenates that are absent in <i>Disc1</i>^Δ2Δ3 mice. (<b>B</b>) Significantly fewer primary neurospheres (P = 0.007; <i>post-hoc</i> Bonferroni p<0.05) derived from dissociated adult hippocampal cells in <i>Disc1</i>^31L/31L mice (n = 6) compared with either wild-type (n = 6) or <i>Disc</i>1^100P/100P (n = 4) mutants. (<b>C</b>) Confocal z-stacks of mouse sections labeled with an antibody raised against the neural progenitor marker Tbr2 (red) and nuclei label Hoechst 33242 (blue) indicate that <i>Disc1</i>^31L/31L mutants (n = 8) have significantly (<b>D</b>) fewer Tbr2 labelled cells (ANOVA, P = 0.014) than either wild-type (n = 9, <i>post-hoc</i> Bonferroni p<0.05) or <i>Disc1</i>^100P/100P (n = 9, <i>post-hoc</i> Bonferroni p<0.05) mice. (<b>E</b>) Cell death as measured by activated caspase-3 immunoreactivity was not significantly different between genotypes (ANOVA, p = 0.74, n = 4 wild type, <i>Disc1</i>^100P/100P; n = 5 <i>Disc1</i>^31L/31L; Scale bar 100 µm. Data presented as mean ± SEM.</p

Loss of immature neurons in <i>Disc1</i>^100P/100P mice.

<p>(<b>A</b>) Immunolabelling of immature neurons residing in the subgranular zone with an antibody raised against doublecortin (DCX) identify frequent gaps (arrows) in DCX staining solely in <i>Disc1</i>^100P/100P mice that reflect a significant loss (<b>B</b>) of DCX⁺ cell bodies compared to wild-type controls (<i>post-hoc</i> Bonferroni p<0.05; n = 8 wild type, n = 5 <i>Disc1</i>^31L/31L. n = 6 <i>Disc1</i>^100P/100P). Data presented as mean ± SEM. Scale Bar (A) 100 µm (B) 25 µm</p

Motor dysfunction in <i>Myk</i>/+ mice.

<p>(A) Gait analysis. <i>Left panel</i>: Mean fore stride and hind stride distance (± SEM) per cm trunk of <i>Myk</i>/+ (<i>n = </i>14) and +/+ (<i>n = </i>17) female mice. There were significant main effects of genotype on fore stride length (<i>F</i>_1,30 = 5.59, <i>P = </i>0.025), hind stride length (<i>F</i>_1,30 = 8.09, <i>P = </i>0.008) and hind stride width (<i>F</i>_1,30 = 24.44, <i>P = </i>0.0001) (left panel). <i>Middle panel</i>: Typical examples of forepaw (red) and hindpaw (blue) placement of <i>Myk</i>/+ and +/+ mice are shown. Scale bar  = 2 cm. <i>Right panel</i>: <i>Myk</i>/+ mouse showing splayed hindlimbs. (B) Balance beam. Mean number of foot slips (left panel) and traversal time (right panel) (± SEM) of <i>Myk</i>/+ (<i>n = </i>26) and +/+ (<i>n = </i>45) mice when traversing a narrow beam 24 hours after training. There were significant main effects of genotype on number of foot slips (<i>F</i>_1,70 = 99.46, <i>P = </i>0.0001) and traversal time (<i>F</i>_1,70 = 43.38, <i>P = </i>0.0001). (C) Tail suspension. Mean hindlimb retraction score (± SEM) of <i>Myk</i>/+ (<i>n = </i>26) and +/+ (<i>n = </i>26) mice suspended by the tail for 30 s. There was a significant main effect of genotype (<i>F</i>_1,51 = 29.00, <i>P = </i>0.0001). Hindlimb retraction is defined as the movement of one of both hindlimbs into the central body axis (photograph). (D) Accelerating rotarod. Mean latency (± SEM) of <i>Myk</i>/+ (<i>n = </i>18) and +/+ (<i>n = </i>21) mice to fall from a rotating rod over three training trials. There were significant main effects of sex (<i>F</i>_1,38 = 9.94, <i>P = </i>0.003) and genotype (<i>F</i>_1,38 = 6.09, <i>P = </i>0.019), but not genotype x sex interaction (<i>F</i>_1,38 = 0.91, <i>P = </i>0.346), females performing better than males regardless of genotype. (E) Tremor. Mean amplitude of displacement (± SEM) of <i>Myk</i>/+ (<i>n = </i>36) and +/+ (<i>n = </i>52) mice across a spectrum of frequencies. There was a significant main effect of genotype on frequency at the maximal amplitude (<i>F</i>_1,87 = 57.1, <i>P = </i>0.0001). *<i>P<</i>0.05; **<i>P<</i>0.01; ****<i>P<</i>0.0001 versus +/+ mice.</p

Thalamocortical, thalamostriatal and intrathalamic functional connectivity in <i>Myk</i>/+ mice.

<p>Summary diagrams showing altered functional connectivity of (A) frontal cortex (FCTX), (B) ventral anterior thalamic nucleus (VAthal), (C) ventromedial thalamic nucleus (VMthal), and (D) ventral posteromedial nucleus (VPMthal) in <i>Myk</i>/+ mice. Only regions where the 95% CI of the VIP exceeded 0.80, in either <i>Myk</i>/+ or +/+ mice, were considered to be functionally connected to the defined “seed” region of interest. The I810N <i>Myshkin</i> mutation-induced alterations in functional connectivity were analysed by permutation test (1000 random permutations of the real data) with significance set at <i>P<</i>0.05. Red denotes a significant increase, whereas blue denotes a significant decrease, in regional functional connectivity in <i>Myk</i>/+ mice relative to +/+.</p

Significant alterations in overt local cerebral glucose utilization in <i>Myk</i>/+ mice.

<p>Data shown as mean ± SEM of the ¹⁴C-2-DG uptake ratio. *denotes <i>P<</i>0.05, **denotes <i>P<</i>0.01 and ***denotes <i>P<</i>0.001 significant genotype effect (Welch's <i>t</i>-test).</p

Cognitive impairment in <i>Myk</i>/+ mice.

<p>(A) Fear conditioning with 1.0-mA footshock. Mean freezing levels (± SEM) of <i>Myk</i>/+ (<i>n = </i>24) and +/+ (<i>n = </i>25) mice at baseline, during training, and in the contextual and cued conditioning tests. There were significant main effects of genotype on freezing in the context test (<i>F</i>_1,48 = 8.52, <i>P = </i>0.005) and in the cue test during presentation of the auditory tone (CS; <i>F</i>_1,48 = 6.20, <i>P = </i>0.016). (B) Conditioned taste aversion. Mean (± SEM) CS consumption scores (intake of saccharin/total fluid) 24 h following pairing with LiCl or saline treatment in <i>Myk</i>/+ and +/+ mice. There were significant main effects of genotype (<i>F</i>_1,47 = 6.51, <i>P = </i>0.014), treatment (<i>F</i>_1,47 = 48.12, <i>P = </i>0.0001), and genotype x treatment interaction (<i>F</i>_1,47 = 4.09, <i>P = </i>0.049). *<i>P<</i>0.05; ***<i>P<</i>0.001; ****<i>P<</i>0.0001 versus +/+ mice.</p

Summary diagram of alterations in brain system functional connectivity and overt alterations in regional cerebral glucose metabolism seen in <i>Myk</i>/+ mice.

<p>Blue shading of neural systems indicates a significant decrease in overt cerebral metabolism while red denotes a significant increase in overt cerebral metabolism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060141#pone-0060141-g004" target="_blank">Figure 4</a>). Blue/broken arrows indicate a decrease in functional connectivity between and within (periaqueductal grey subfields) neural systems in <i>Myk</i>/+ mice relative to +/+ littermates. Red/solid arrows indicate increased functional connectivity between and within (thalamic nuclei) neural systems in <i>Myk</i>/+ mice relative to +/+ littermates.</p