Primers used for quantification of mRNA expression.

<p>Primers used for quantification of mRNA expression.</p

Inactivation of neuropsin reduces the impairment of spatial memory performance following chronic stress or elevated corticosterone.

<p>(A) There is no significant difference in the water maze training curve between KO and WT mice. In the chronic stress groups, there is also no difference in the training curve. (B) Two weeks later, there is no significant difference in memory as measured by time spent in the target zone and the number of phantom platform crosses; however in the chronic stress group, neuropsin KO mice spent more time in the target zone and made a greater number of phantom platform crosses. (C) In both oil and corticosterone injection groups (20 mg/kg/day) there is no difference in the training curves. (D) KO mice performed significantly better in memory tests that WT following corticosterone injection. n = 10–11 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Neuropsin-deficient mice are resistant to chronic stress or elevated corticosterone induced depressive-like behaviour.

<p>(A, C, E) There is no significant difference between WT and KO mice in the novelty suppressed feeding, forced swim or sucrose preference tests without prior exposure to stress. After chronic stress, neuropsin KO mice approach food pellets more quickly than WT mice in the novelty suppressed feeding test. In the forced swim test, the immobility time of KO mice is shorter than WT mice. KO mice drink more sucrose than WT mice in the sucrose preference test. (B, D, F) There is no significant difference between WT and KO mice in the oil injection group. After chronic corticosterone injection (20 mg/kg/day), neuropsin KO mice show less depressive-like behaviour in novelty suppressed feeding, forced swim test, and sucrose preference test than WT mice. n = 8–10 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Neuropsin-deficient mice are partially protected against the effect on hippocampal neurogenesis elicited by chronic stress or corticosterone injection.

<p>(A) Chronic stress (n = 7–10) or chronic corticosterone injections (n = 6) dramatically decrease hippocampal neurogenesis. Neuropsin KO mice have a higher cell count of DCX positive cells in the dentate gyrus. (B) There are more BrdU positive cells in the dentate gyrus in KO mice than WT mice. (C) There is no significant difference in KI67 cell counts in both groups; n = 7–10 for chronic stress groups, n = 6 for chronic corticosterone groups. Scale bar = 200μm, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Neuropsin interacts with corticosterone to influence dendritic spine density and myelination.

<p>(A, B) The hippocampal dendritic spine density decreases in both WT and KO mice following chronic corticosterone injections but KO mice preserve more dendritic spines compared to WT mice, n = 4, Scale bar = 10μm. (C, D) Immunohistochemistry staining of CNPase in cortex and hippocampus. Scale bar = 200μm. (E, F) After chronic corticosterone injections, KO mice retain a higher expression of <i>Cnp</i> and <i>Mog</i> compared to WT mice in the hippocampus but there is no significant difference in the cortex. n = 6 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Neuropsin-deficient mice are resistant to glutamate transmission dysregulation induced by chronic elevated plasma corticosterone.

<p>(A) Gene expressions of <i>Klk8</i>, <i>GR</i>, <i>Sgk1</i> and <i>Fkbp5</i> in the hippocampus in WT and KO mice after chronic corticosterone or oil injection. (B) Gene expressions of glutamate receptors, <i>GluN2A</i>, <i>GluN2B</i> and <i>Camk2b</i> and glutamate reuptake (EAAT1), vesicle packing (VGluT1) in hippocampus. (C) Detection of NADPH induced reactive oxygen species in the hippocampus of WT and KO mice after chronic corticosterone or oil injection. n = 9–13 in each group. (D-I) Protein expressions of GR, SGK1, Fkbp5, NR2b, EAAT1 and CamKII measured by western blot assay. n = 6 in each group, values represent mean ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Inactivation of neuropsin blocks corticosterone induced hippocampal neuronal activity.

<p>(A) One day after chronic corticosterone or oil injection in WT and KO mice, c-Fos positive cells in the dentate gyrus (DG), CA1 and CA3. (B-D) Quantification of c-Fos positive cells in the DG, CA1, and CA3. KO/CORT mice display fewer c-Fos positive cells in the CA1 and CA3 compared to WT/CORT mice. n = 6 in each group, scale bar = 200 μm, values represent mean ± SEM. * <i>p</i> < 0.05, *** <i>p</i> < 0.001.</p

Antidepressant-like behavior in mice engaged in voluntary exercise for 28 days (n = 8).

<p>A series of antidepressant-like behavioral tests were conducted. (A) There was no significant difference in the activity in the open field test. (B) Exercise group showed lower levels of digging activity in a new cage with a 5 cm layer of sawdust bedding. (C) In the novelty suppressed feeding test, the exercise group increased the time spent in eating in a rat odor cage compared to controls. No significant differences between the groups in food consumption at the home cage. (D) In the novelty induced hypophagia test, the exercise group spent more time drinking sweet milk under bright lighting cage condition, but no significant differences were observed under dim lighting cage conditions. Values are means ± SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

Validation by real-time PCR of three genes that show differential expression in the microarray analysis.

<p>The expression of Nptx2, Bdnf and Plekha2 is shown for the fluoxetine and exercise groups relative to control. (A) Relative expression based on (A) expression arrays and (B) real-time PCR. Values are means ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Hippocampal gene expression profiles in response to different antidepressant treatments.

<p>(A) Changes in expression following exercise compared with the changes in the fluoxetine treated group. Each point is the average expression level of the treated group divided by the average expression level in the control group for one of the 87 genes showing significant differential expression. The Y and X axes are on a log scale, base 2. The line is the best fit linear regression. (B) Changes in expression in an enriched environment as a function of the changes in the fluoxetine treated group. (C) Changes in expression for 36 genes (out of 87) with the highest fold change following chronic fluoxetine treatment compared to controls. (D) Comparison of the effect of fluoxetine on gene expression in the current study with the results obtained by Miller et al. (2008) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035901#pone.0035901-Miller1" target="_blank">[17]</a>.</p