Effects of CUS on neuritin expression, determined by in situ hybridization and quantitative real-time (RT) PCR. (A) Rats were exposed to CUS for 35 d and received either saline or fluoxetine (CUS + FLX) for the last 21 d, and sections were subjected to quantitative in situ hybridization. Representative autoradiograms from hippocampal sections from in situ hybridization are shown for home cage control (CTR), CUS, CUS + FLX-, and FLX-treated animals (n = 5 per group). (B) Quantified expression of neuritin mRNA from the indicated subregions in A. Results are expressed as a ratio of CTR and are the mean ± SEM, each analyzed in duplicate brain sections. Neuritin mRNA was decreased in CUS animals compared with CTR animals (CA1: F_{3, 15} = 9.27, **P < 0.01; CA3: F_{3, 15} = 13.45, **P < 0.001; DG: F_{3, 15} = 11.96, **P < 0.001). CUS animals injected with FLX showed an increase in neuritin mRNA compared with CUS animals (CA1: P = 0.011; CA3: P = 0.04; DG: P = 0.006). FLX in the nonstressed CTR did not increase neuritin mRNA compared with CTR (CA1: P = 0.075; CA3: P = 0.062; DG: P = 0.078). In the cortex, there were no differences between groups (P > 0.2). **P < 0.01 compared with CTR and ^#P < 0.05 and ^##P < 0.01 compared with CUS (two-way ANOVA, Fisher’s LSD post hoc analysis). (C) Rats were exposed to CUS and FLX as described above, and whole hippocampus was subjected to quantitative RT-PCR analysis. Results are normalized to cyclophilin and expressed as the mean ratio of fold change ± SEM of six individual animals. *P < 0.05; ***P < 0.001, two-way ANOVA.

AAV-Nrn infusions into the hippocampus produce antidepressant behavioral actions. (A) Experimental design. Animals were injected with AAV-ctl or AAV-Nrn; 5 wk later, they were tested in behavioral paradigms, and hippocampal sections were then harvested for in situ hybridization (ISH) and immunohistochemistry (IHC). (B) NSFT. A significant decrease in the latency to feed was shown in AAV-Nrn–injected animals compared with AAV-ctl–injected animals [t₍₁₉₎ = 2.57, *P < 0.05]. (C) FST. AAV-Nrn–injected rats had a shorter immobility score (time in seconds) than AAV-ctl–injected rats [t₍₁₉₎ = 4.14, ***P < 0.001]. (D) Home cage feeding. There was no difference in the home cage food intake between AAV-ctl–injected and AAV-Nrn–injected rats [t₍₃₀₎ = 0.35, P = 0.73]. (E) LMA. The total distance moved in the box was similar between groups [t₍₁₉₎ = 0.17, P > 0.5]. Results are the mean ± SEM averaged from AAV-ctl–injected (n = 10) vs. AAV-Nrn–injected (n = 11) rats. Student t test.

Neuritin knockdown causes depressive behaviors. (A) Experimental design. Lenti-shNrn was infused into the hippocampal DG, and behavioral testing was initiated 5 wk later. (B) Knockdown efficiency of lenti-shNrn. Lenti-shNrn was transfected into rat primary hippocampal neuronal cells, and neuritin and β-actin mRNA levels were measured by real-time (RT) PCR. Treatment of cells with BDNF (10 ng/mL) for 6 h induced neuritin mRNA expression, and the increase was reduced by lenti-shNrn in vitro in a concentration-dependent manner. (C) Knockdown efficiency of lenti-shNrn. Quantitative RT-PCR of neuritin mRNA in microdissected DG from hippocampal sections taken from lenti-ctl–injected and lenti-shNrn–injected animals confirms that neuritin knockdown decreases neuritin mRNA in the DG (***P < 0.001). (D) NSFT. Lenti-shNrn infusion increased the latency to feed compared with lenti-EGFP–injected control animals (*P < 0.05, Student t test). (E) FST. Animals injected with lenti-shNrn had higher immobility times than those injected with lenti-EGFP when assessed by means of a 15-min test (***P < 0.001, Student t test). (F) SPT. Lenti-shNrn decreased sucrose preference compared with lenti-EGFP–injected animals (*P < 0.05, Student t test). (G) Home cage feeding. There was no difference in the amount in the home cages between groups (P = 0.8). (H) LMA. Lenti-shNrn did not influence LMA (P = 0.16, Student t test). Results are the mean ± SEM averaged from seven animals per group. (I) FST. (Left) Experimental design. Two-way ANOVA, main effect of virus: F_{3, 21} = 25.79, P < 0.001; main effect of BDNF: F_{3, 21} = 16.50, P < 0.001; higher immobility time by lenti-shNrn (*P < 0.05). BDNF-infused rats had a shorter immobility score than lenti-ctl–injected rats (**P < 0.01) and the effect of BDNF on immobility was blocked by lenti-shNrn (***P < 0.001). Scores are measured by means of a 15-min test. Results are the mean ± SEM averaged from seven animals per each group.

Neuritin increases dendritic arborization and synaptogenesis. (A) In situ hybridization analysis was conducted to examine the expression of neuritin mRNA, and representative autoradiograms are shown for AAV-ctl and AAV-Nrn. (B) GFP staining is also shown and further demonstrates localization of AAV infection of the DG. (C) Representative images of GFP(+) cells from AAV-ctl– and AAV-Nrn–injected animals. A continuous stretch of dendritic shaft localized in the outer half of the granular layer was identified in the outer molecular layer and imaged. (D) Immunocytochemical analysis showing that the dendritic arborization was increased in AAV-Nrn–injected rats compared with AAV-ctl–injected rats (***P < 0.001). The data were expressed as the number of bifurcations per GFP(+) neurons (n = 26 cells and n = 16 cells for AAV-ctl– and AAV-Nrn–injected groups, respectively). (E) Representative images are shown of high-magnification Z-stack projections of apical tuft segments of GFP(+) DG granule cells from AAV-ctl– and AAV-Nrn–injected rats. (F) Density of dendritic spines was significantly increased in AAV-Nrn–injected rats compared with AAV-ctl–injected rats. (***P < 0.001) (n = 10 neurons from 6 rats in each group). The data were expressed as the number of spines per 10 μm. (G) Spine head diameter of mushroom spines was increased in AAV-Nrn–injected rats compared with AAV-ctl–injected rats (***P < 0.001) (n = 12–15 neurons from 6 rats in each group). Results are the mean ± SEM. (Scale bars: A, 200 μm; B, 50 μm; C and E, 10 μm.) Student t test.

AAV-Nrn infusions into the hippocampus block the behavioral deficits caused by CUS exposure. (A) Experimental design. Rats were injected with AAV-ctl (n = 16) or AAV-Nrn (n = 16). Each of the virus-infected cohorts was split into two experimental groups and exposed to home cage or CUS for 21 d. The efficacy of neuritin overexpression and CUS on behavioral performances of the animals was measured for 3–4 consecutive days starting on day 40 (D40). (B) SPT. Two-way ANOVA revealed that there was a main effect of virus (F_{1, 28} = 5.91, P < 0.05), a main effect of stress (F_{1, 28} = 18.92, P < 0.001), and interaction (F_{1, 28} = 8.8, P < 0.01). Further analysis indicates that CUS decreased sucrose preference compared with home cage in AAV-ctl–injected animals (^###P < 0.001). Neuritin increased sucrose preference in CUS rats (***P < 0.001) but not in home-caged rats (P = 0.73). There was no difference in AAV-Nrn–injected CUS animals and AAV-ctl–injected home cage animals (P = 0.13). (C) Active avoidance test (AAT). Two-way ANOVA: main effect of virus, F_{1, 28} = 7.19, P < 0.05; main effect of stress, F_{1, 28} = 5.37, P < 0.05; interaction F_{1, 28} = 8.19, P < 0.01. Further analysis indicates that CUS-exposed rats had more escape failures than home cage rats in AAV-ctl animals (^##P < 0.01) and that neuritin overexpression decreased escape failures only in CUS rats (**P < 0.01) but not in nonstressed rats (P = 0.87). There was no difference in AAV-Nrn–injected CUS animals and AAV-ctl–injected home cage animals (P = 0.74). (D) Representative images of high-magnification Z-stack projections of apical tuft segments of GFP(+) DG granule cells. (E) Density of dendritic spines. Two-way ANOVA: main effect of virus, F_{3, 42} = 19.05, P < 0.001; main effect of stress, F_{3, 42} = 7.45, P < 0.01; interaction F_{3, 42} = 0.001, P > 0.05. The density of dendritic spines was significantly decreased in CUS rats (*P < 0.05), and the effects were blocked in AAV-Nrn–injected rats (**P < 0.01). n = 10–19 GFP(+) neurons from four rats in each group. The data were expressed as the number of spines per 10 μm. (Scale bar: 10 μm.)

AAV-Nrn infusion into the hippocampus improves hippocampal-dependent memory. (A) Rats were injected with AAV-ctl or AAV-Nrn and then tested in object recognition 30 d later. Both groups show comparable exploration of the novel object at 1.5 h after training (familiar vs. novel object, P < 0.05; t < 1.00, P = 0.769). The discrimination index was not different between the two groups (t < 1.00, P = 0.427). (B) In a separate experiment (45 d after virus infusion), where testing was conducted 24 h after training, rats injected with AAV-Nrn show significantly higher levels of discrimination between the familiar and novel objects [unpaired t test, familiar vs. novel object: t₍₁₂₎ = 3.47, **P < 0.005]) compared with AAV-ctl (familiar vs. novel object: t₍₁₂₎ = 0.871, P = 0.4). The discrimination index was significantly higher in animals injected with AAV-Nrn than in controls [t₍₁₂₎ = 4.76, ***P < 0.001]. (C) Fear conditioning (FC). The freezing behavior of animals treated with AAV-ctl and AAV-Nrn (35 d after virus infusion) is shown. On the training day, both groups showed similar levels of freezing in the context after each single 2-s, 0.55-mA foot shock was delivered (P > 0.05). (D) FC, contextual test. AAV-Nrn animals exhibit significantly higher levels of freezing when reexposed to the fear-conditioned context 24 h after training [t₍₁₄₎ = 2.14, *P < 0.05]. Results are presented as the mean ± SEM averaged from seven animals per each group. Unpaired t-test.