1. Volume 148, Issue 5, Pages 1051-1064 (March 2012)
Interplay between DISC1 and GABA Signaling Regulates Neurogenesis in Mice and Risk for Schizophrenia 
Ju Young Kim, Cindy Y. Liu, Fengyu Zhang, Xin Duan, Zhexing Wen, Juan Song, Emer Feighery, Bai Lu, Dan Rujescu, David St Clair, Kimberly Christian, Joseph H. Callicott, Daniel R. Weinberger, Hongjun Song, Guo-li Ming 
Cell 
Volume 148, Issue 5, Pages (March 2012)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
DISC1 KD-Induced Precocious Dendritic Growth of Newborn Neurons in the Adult Dentate Gyrus Requires GABA-Induced Depolarization
(A) A schematic diagram of the experimental design and sample projected confocal images of newborn neurons with retrovirus-mediated coexpression of GFP and a control shRNA (C1), shRNA-DISC1 (D1), or shRNA-NKCC1 (NK1) in the adult mouse dentate gyrus at 14 dpi. The scale bar represents 50 μm.
(B) Summary of total dendritic length of GFP+ neurons under different conditions. Values represent mean ± SEM (n = 4 animals with a total of 29–36 neurons for each condition; ∗p < 0.05; ANOVA).
(C–F) Effect of retrovirus-mediated double NKCC1 and DISC1 KD on dendritic development and AKT/mTOR signaling in newborn neurons in the adult dentate gyrus. Shown in (C) are schematic diagrams of retroviral vectors and experimental design, and sample projected confocal images of a newborn neuron with double KD at 14 dpi. Shown are summaries of total dendritic length (D) and quantifications of pAKT (E) and pS6 (F) levels in the cytosol of new neurons at 14 dpi. Numbers associated with the bar graph represent total numbers of neurons examined under each condition. Values represent mean ± SEM (n = 4 animals for each condition; ∗p < 0.05; ANOVA).
See also Figure S1.
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4. Figure 2
DISC1 KD-Induced Precocious Dendritic Development of Newborn Neurons in the Adult Dentate Gyrus Is Enhanced by Activation of GABA Signaling
(A–D) Effect of vigabatrin (VGA) on dendritic development and AKT/mTOR signaling in newborn neurons in the adult hippocampus with or without DISC1 KD. Shown in (A) are the schematic diagram of the experimental design and sample projected confocal images of new neurons with retrovirus-mediated coexpression of GFP and a control shRNA (C1) or shRNA-DISC1 (D1), with or without VGA injection (25 μg/g body weight per day from 3 to 6 dpi, i.p.) and examined at 7 dpi. The scale bar represents 50 μm. Shown are summaries of total dendritic length (B) and quantifications of pAKT (C) and pS6 (D) levels in the cytosol of newborn neurons at 7 dpi. Values represent mean ± SEM (n = 4 animals; ∗p < 0.05; n.s., p > 0.1; ANOVA).
(E–H) Same as in (A)–(D), except that pentobarbital (25 μg/g body weight per day from 6 to 10 dpi; i.p.) was injected and analysis was performed at 11 dpi.
(H–K) Same as in (B)–(D), except that newborn neurons expressing shRNA-control (C1), KIAA1212 (K1), shRNA-NK1 (NK1), or both K1 and NK1 were analyzed at 14 dpi.
(L) A model of interaction between depolarizing GABA and DISC1 signaling in regulating dendritic growth of newborn neurons in the adult dentate gyrus.
See also Figure S2.
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5. Figure 3
DISC1 KD Fails to Affect Development of Newborn Dentate Granule Neurons during Early-Postnatal Hippocampal Neurogenesis
(A) A schematic diagram of the experimental design and sample projected confocal images of newborn neurons with retrovirus-mediated coexpression of GFP and a control shRNA (C1), or shRNA-DISC1 (D1), in the dentate gyrus of neonatal mouse hippocampus. The scale bar represents 50 μm.
(B–D) Summary of the mean total dendritic length (B), soma size (C), primary dendrite number (D) of GFP+ neurons under different conditions. The same data on the time course of dendritic development of shRNA-C1/GFP+ neurons in the adult hippocampus (P42) as in Figure 1B are replotted in (B) for direct comparison. Values represent mean ± SEM (n = 4–6 animals with a total of 36–45 neurons for each condition; p > 0.05; ANOVA).
(E) Positioning of newborn neurons in the dentate gyrus under different conditions. Shown is the summary of the distribution of soma of GFP+ neurons within the four domains in the dentate gyrus (as defined in Figure S1C). The same group of neurons as in (B)–(D) was used.
See also Figure S3.
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6. Figure 4
Newborn Neurons during Adult and Early-Postnatal Neurogenesis Exhibit Differential Tempo in Shifting the Polarity of GABA Responses
(A) A schematic diagram of the experimental design and sample Ca2+ imaging analysis of new neurons expressing RFP and shRNA-C1 in P42 animals. Sample confocal images of RFP and Ca2+ dye Fluo4-AM, as well as Ca2+ responses were shown at the basal level, followed by application of GABAAR agonist muscimol (musc.; 10 μM), and then Ca2+ ionophore ionomycin (iono.; 10 μM). White arrowheads point to RFP+Fluo4+ newborn cells. The scale bar represents 20 μm.
(B) Summary of mean peak Ca2+ responses to muscimol for newborn neurons expressing shRNA-C1 (C1), shRNA-NKCC1 (NK1), or shRNA-D1 (D1) in the adult dentate gyrus. The values of Ca2+ responses are normalized to the mean fluorescence intensity measured at the baseline condition (set as 0%) and after the ionomycin treatment (set as 100%) in the same cell. Values represent mean ± SEM (n = 6–7 cells; ∗p < 0.01; ANOVA).
(C and D) Same as (A) and (B), except that retroviruses coexpressing RFP and shRNA-C1 (C1), shRNA-KCC2 (K2), or shRNA-D1 (D1) were injected into P10 animals.
See also Figure S4.
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7. Figure 5
DISC1 KD Induces Precocious Dendritic Development during Early-Postnatal Neurogenesis in the Presence of Extended Period of GABA-Induced Depolarization
(A) A schematic diagram of the experimental design.
(B) Sample confocal images of newborn neurons after stereotaxic injection of a mixture of retroviruses coexpressing shRNA-DISC1 (D1)/DsRed and those coexpressing shRNA-KCC2 (K2)/GFP into the dentate gyrus of the P10 animal and analyzed at 7 dpi. The scale bar represents 20 μm.
(C and D) Summary of the mean total dendritic length of newborn neurons at 7 dpi under different conditions. Rapamycin (20 mg/kg body weight) or saline was i.p. injected daily after viral injection in (D). Values represent mean ± SEM (n = 6–8 animals with a total of 31–50 neurons for each condition; ∗p < 0.05; ANOVA).
(E and F) Summary models of interaction between DISC1 and depolarizing GABA in regulating dendritic development in a context-dependent fashion. Shown in (E) is a schematic diagram of interaction between depolarizing GABA and DISC1 signaling in regulating dendritic development. Shown in (F) is a model of DISC1 signaling in regulating neuronal development during early-postnatal and adult hippocampal neurogenesis and its temporal constraints.
See also Figure S5.
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8. Figure 6
Maternal Deprivation Stress Synergizes with DISC1 KD in Regulating Early-Postnatal Hippocampal Neurogenesis
(A) A diagram of the experimental design.
(B) Sample confocal images of new neurons at 7 dpi after retrovirus-mediated expression of control shRNA (C1), shRNA-DISC1 (D1), shRNA-NKCC1 (NK1), or coexpression of D1 and NK1, in P10 dentate gyrus with or without maternal deprivation stress. The scale bar represents 50 μm.
(C–F) Summaries of mean total dendritic length (C and E) and Sholl analysis (D and F) of new neurons at 7 dpi in the early-postnatal dentate gyrus. Rapamycin (Rapa; 20 mg/kg body weight) was i.p. injected daily after viral injection for some animals as indicated. Values represent mean ± SEM (n = 4–8 animals; ∗p < 0.01; ANOVA).
See also Figure S6.
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9. Figure 7
DISC1 and NKCC1 Interact Epistatically to Affect Risk for Schizophrenia
(A) Haploview LD plot (R-squared; based on the Scottish sample) of SNPs on the Illumina chip in DISC1 showing interactions with SNPs in NKCC1.
(B) Linkage disequilibrium (R-squared measure) of SNPs in NKCC1 (HapMAP CEU data) showing two SNPs on the Illumina chip that were used for the interaction analysis with DISC1.
(C) Interaction between rs10089 (NKCC1) and rs (DISC1) and risk for schizophrenia in three independent case-control samples of European ancestry.
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10. Figure S1
Effects of Genetic Manipulation of DISC1 or Depolarizing GABA Signaling on the Development of Newborn Neurons in the Adult Dentate Gyrus, Related to Figure 1
(A and B) The effect of DISC1 KD and NKCC1 KD on soma size (A) and number of primary dendrites (B) of newborn neurons in the dentate gyrus of the adult hippocampus. Retroviruses co-expressing GFP and control shRNA (C1), shRNA-DISC1 (D1), or shRNA-NKCC1 (NK1), or a mixture of retroviruses co-expressing GFP and NK1 and those co-expressing DsRed and D1, were stereotaxically injected into the dentate gyrus of adult mice at P42 and newborn neurons were analyzed at 7, 11 and 14 dpi, respectively. The same groups of neurons as in Figures 1B and 1D were analyzed. Values represent mean ± SEM (n = 4 animals with a total of neurons for each condition; ∗: p < 0.05, ANOVA).
(C) Summary of soma positioning of newborn neurons in the adult dentate gyrus under different conditions. Shown on top is a schematic diagram of dentate gyrus divided into four subdomains: inner granule cell layer (area 1), middle granule cell layer (area 2), outer granule cell layer (area 3) and molecular layer (area 4). Shown at the bottom is the summary of the cell body distribution of newborn neurons within each subdomain for each condition. The same groups of neurons as in Figure 1B were analyzed. Values represent mean (n = 4 animals).
(D) Sholl analysis of dendritic complexity of newborn neurons in the adult hippocampaus at 7 and 14 dpi. The same groups of neurons as in Figure 1B were analyzed. Values represent mean ± SEM.
(E and F) In vitro analysis of the efficacy of shRNAs against mouse γ2 subunit of GABAARs. The expression construct for mouse γ2 subunit of GABAARs and shRNA vector (pUEG), or shRNA-γ2 were transfected into HEK293 cells. After 48 hr, cell lysates were processed for Western blot analysis for γ2 subunit and then replotted for GAPDH. Shown in (E) is a sample West Blot and in (F) is the quantification of the efficacy of two different shRNAs. Values represent mean ± SEM (n = 3; ∗: p < 0.05; ANOVA).
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11. Figure S2
Interaction between GABA and DISC1 Signaling in Regulation of Soma Size, Primary Dendrite Numbers and Positioning of New Neurons in the Adult Dentate Gyrus, Related to Figure 2
(A–C) Effect of vigabatrin (VGA) treatment on the development of newborn neurons expressing shRNA-C1 (C1) or shRNA-D1 (D1) in the adult dentate gyrus. Shown are summaries of the mean soma size (A), number of primary dendrites (B) and distribution of soma within each subdomains in the dentate gyrus (C). The same groups of neurons as in Figure 2B were analyzed. Values represent mean ± SEM (n = 4 animals with a total of neurons for each condition).
(D–F) Effect of pentobarbital (Pento) treatment on the development of newborn neurons expressing C1 or D1 in the adult dentate gyrus. Similar as in (A)–(C), except that the same groups of neurons as in Figure 2F were analyzed.
(G–I) Effect of shRNA-NK1 and KIAA1212 expression on the development of newborn neurons in the adult dentate gyrus. Similar as in (A)–(C), except that the same groups of neurons as in Figure 2I were analyzed.
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12. Figure S3
Effects of DISC1 KD on Newborn Neuron Development during Early-Postnatal Hippocampal Neurogenesis, Related to Figure 3
(A) Dendritic development of newborn neurons during early-postnatal hippocampal neurogenesis. Sholl analysis of dendritic complexity of newborn neurons at 3, 7, 11, and 14 dpi after retroviral injection into the dentate gyrus of the P10 animal. The same groups of neurons as in Figure 3 were used. Values represent mean ± SEM (n = 4-6 animals with a total of neurons for each condition).
(B) Neuronal subtype differentiation of neural progenitors during early-postnatal hippocampal neurogenesis. Shown are sample confocal images of immunostaining of Prox-1, a dentate granule cell maker, and Parvalbumin, an interneuron subtype marker, for retrovirally labeled newborn neurons co-expressing GFP and shRNA-C1 or shRNA-D1 at 7 dpi. The scale bar represents 25 μm.
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13. Figure S4
In Vitro Analysis of the Efficacy of shRNAs against Mouse kcc2, Related to Figure 4
(A) Sample Western blot analysis of KCC2 and GAPDH. The expression construct for mouse KCC2 and shRNA vector (pUEG), or shRNA-KCC2 were transfected into 293 cells. After 48 hr, cell lysates were processed for Western blot analysis for KCC2 and then replotted for GAPDH.
(B) Quantification of Western Blot analysis. Shown is a summary of quantitative analysis of the efficacy of three different shRNAs against mouse kcc2 in vitro. Values represent mean ± SEM (n = 3). shRNA-KCC2-2 (shRNA-K2) was subsequently used for functional analysis in Figures 4 and 5.
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14. Figure S5
Effects of DISC1 and KCC2 KD on New Neuron Development during Early-Postnatal Hippocampal Neurogenesis, Related to Figure 5
Retroviruses expressing control shRNA (C1), shRNA-DISC1 (D1), or shRNA-KCC2-2 (K2), or co-expressing of D1 and K2, were stereotaxically injected into dentate gyrus of the neonatal mouse at P10 and newborn neurons were analyzed at 7 dpi for sholl analysis of dendritic growth (A and E), soma size (B and F), number of primary dendrites (C and G) and neuronal positioning (D and H). The same groups of neurons as in Figures 5C and 5F were analyzed.
Cell  , DOI: ( /j.cell )
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15. Figure S6
Effect of Maternal Deprivation Stress on Newborn Neurons with DISC1 KD during Early-Postnatal Hippocampal Neurogenesis, Related to Figure 6
(A) Summary of the mean peak Ca2+ responses to muscimol for newborn neurons at 7 dpi after maternal deprivation or sham treatment. Same as in Figure 4, values of Ca2+ responses are normalized to the mean fluorescence intensity measured at the baseline condition (set as 0%) and after the ionomycin treatment (set as 100%) in the same cell. The same data for newborn neurons under the normal condition from Figure 4D is reploted for direct comparison. Values represent mean ± SEM (n = 6-7 cells; ∗: p < 0.01; ANOVA).
(B and C) Summary of soma size (B) and number of primary dendrites (C) for newborn neurons in early-postnatal dentate gyrus expressing shRNA-C1 (C1), shRNA-D1 (D1), shRNA-NKCC1 (NK1), or both D1 and NK1, at 7 dpi after maternal deprivation stress or sham treatment. The same groups of neurons as in Figure 6C were used.
(D) Positioning of newborn neurons in the dentate gyrus under different conditions. Shown are sample confocal images of DAPI and newborn neurons expressing C1, D1, NK1, or co-expressing D1/DsRed and NK1/GFP at 7 dpi. The scale bar represents 50 μm. Also shown is the summary of the distribution of soma of newborn neurons within the four domains in the dentate gyrus (as defined in Figure S1C). The same group of neurons as in (B) was used.
(E–H) Effect of rapamycin treatment on new neuron development during early-postnatal neurogenesis. Shown in (E) is the confirmation of the efficacy of rapamycin (Rapa) on the reduction of pS6 levels with Western Blot analysis. Also shown are summaries of soma size (F), number of primary dendrites (G), and neuronal positioning (H) of the same group of neurons in Figure 6E.
Cell  , DOI: ( /j.cell )
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