Adolescent but not adult-born neurons are critical for susceptibility to chronic social defeat

Recent evidence implicates adult hippocampal neurogenesis in regulating behavioral and physiologic responses to stress. Hippocampal neurogenesis occurs across the lifespan, however the rate of cell birth is up to 300% higher in adolescent mice compared to adults. Adolescence is a sensitive period in development where emotional circuitry and stress reactivity undergo plasticity establishing life-long set points. Therefore neurogenesis occurring during adolescence may be particularly important for emotional behavior. However, little is known about the function of hippocampal neurons born during adolescence. In order to assess the contribution of neurons born in adolescence to the adult stress response and depression-related behavior, we transiently reduced cell proliferation either during adolescence, or during adulthood in GFAP-Tk mice. We found that the intervention in adolescence did not change adult baseline behavioral response in the forced swim test, sucrose preference test or social affiliation test, and did not change adult corticosterone responses to an acute stressor. However following chronic social defeat, adult mice with reduced adolescent neurogenesis showed a resilient phenotype. A similar transient reduction in adult neurogenesis did not affect depression-like behaviors or stress induced corticosterone. Our study demonstrates that hippocampal neurons born during adolescence, but not in adulthood are important to confer susceptibility to chronic social defeat

Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase α3 missense mutant mice

Missense mutations in ATP1A3 encoding Na(+),K(+)-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+),K(+)-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na(+),K(+)-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+),K(+)-ATPase α3, including upon the K(+) pore and predicted K(+) binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+),K(+)-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC

Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

Missense mutations in ATP1A3 encoding Na(+),K(+)-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+),K(+)-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na(+),K(+)-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+),K(+)-ATPase α3, including upon the K(+) pore and predicted K(+) binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+),K(+)-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC

Ablation of proliferating neural stem cells during early life is sufficient to reduce adult hippocampal neurogenesis

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Deficits in social behavioral tests in a mouse model of alternating hemiplegia of childhood

Social behavioral deficits have been observed in patients diagnosed with alternating hemiplegia of childhood (AHC), rapid-onset dystonia-parkinsonism and CAPOS syndrome, in which specific missense mutations in ATP1A3, encoding the Na+, K+-ATPase α3 subunit, have been identified. To test the hypothesis that social behavioral deficits represent part of the phenotype of Na+, K+-ATPase α3 mutations, we assessed the social behavior of the Myshkin mouse model of AHC, which has an I810N mutation identical to that found in an AHC patient with co-morbid autism. Myshkin mice displayed deficits in three tests of social behavior: nest building, pup retrieval and the three-chamber social approach test. Chronic treatment with the mood stabilizer lithium enhanced nest building in wild-type but not Myshkin mice. In light of previous studies revealing a broad profile of neurobehavioral deficits in the Myshkin model – consistent with the complex clinical profile of AHC – our results suggest that Na+, K+-ATPase α3 dysfunction has a deleterious, but nonspecific, effect on social behavior. By better defining the behavioral profile of Myshkin mice, we identify additional ATP1A3-related symptoms for which the Myshkin model could be used as a tool to advance understanding of the underlying neural mechanisms and develop novel therapeutic strategies

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

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

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

Structural modelling of Na⁺,K⁺-ATPase α3 mutations.

<p>(A) Na⁺,K⁺-ATPase α3 wild-type (left), the I810S mutant (AHC; centre) and the I810N mutant (<i>Myshkin</i>; right). (B) Na⁺,K⁺-ATPase α3 wild-type (left), the I274N mutant (AHC; centre) and the I274T mutant (RDP; right). Side chain contact between Δ272 and Δ274 at the cytoplasmic end of the K⁺ pore is shown in yellow for the wild-type protein and the I274T mutant, but this contact is lost in the I274N mutant. (C) Na⁺,K⁺-ATPase α3 wild-type (left), the D801N mutant (AHC; centre) and D801Y mutant (RDP; right). In the D801N mutant, the electrostatic interaction at Δ801 with both K⁺ ions is lost due to replacement of terminal oxygen with nitrogen, resulting in the obstruction of the K⁺ pore, likely to markedly affect conductance rates through the pore, while the interaction of K⁺2 with E776 is maintained. In the D801Y mutant, there is a predicted loss of interaction of K⁺2 with E776. (D) Na⁺,K⁺-ATPase α3 wild-type (left), the D923Y mutant (AHC; centre) and the D923N mutant (RDP; right).</p