Figure 1.
AAV-hGATA-enhanced GFP (eGFP) vector construction and expression in the hippocampal neurons.
(a) RT-PCR analysis demonstrating expression of rGATA-2, but not r-GATA-1 in hippocampus. (b) Quantitative RT-PCR analysis of rGATA-2 in the rat hippocampus exposed to acute stress or CUS in the presence or absence of ketamine treatment. Rats were exposed to acute restraint stress for 2 h or CUS for 21 d and received either saline or ketamine (Stress+KET), and sections were subjected to quantitative RT-PCR. Results are expressed as a ratio of nonstressed controls and are the mean ± SEM, each analyzed in triplicate brain sections. rGATA-2 mRNA was increased in acute and CUS animals compared with nonstressed animals (P<0.01). CUS animals injected with KET showed a decrease in rGATA-2 mRNA compared with CUS animals (P<0.05; n = 6 animals in each group). (c) L-ITR and R-ITR, left and right inverted terminal repeats, respectively; CMV, cytomegalovirus promoter. (d) RT-PCR of cDNA isolated from cultured hippocampal neurons transfected with AAV-hGATA1 and AAV-hGATA2. (e) Western blotting analysis was conducted to examine the expression of hGATA transcription factors, and representative blots were shown. The abundance of hGATA-1 (43 kD) and hGATA-2 (51 kD) protein in the nuclear extracts is increased in response to infection with AAV-hGATA1 or AAV-hGATA2. Nuclear extracts were determined by lamin B1, a nuclear marker. (f) Images of hippocampal neurons infected with AAV and stained with antibodies to GFP and MAP2. (g) The total number of cells was revealed by nuclear staining with DAPI. Transfection with AAV-hGATA1 or AAV-hGATA2 showed the same fraction of GFP(+) cells colabeled with MAP2 compared with transduction with AAV-ctl (P>0.05). The percentage of MAP2(+) cells among GFP(+) cells is shown as a fraction of the total GFP number of cells. Total MAP2(+) and GFP(+) cells (arrowheads) were counted with a microscope in 10 non-overlapping fields per well. Values represent mean ± s.e.m. from five independent experiments. Scale bar, 50 µm.
Figure 2.
hGATA1 and hGATA-2 decreases dendritic arborization and spine density.
Neuronal complexity in the hippocampal neurons transfected with AAV-hGATA1 or AAV-hGATA2 was analyzed based on three-dimensional projections of confocal stack images through complete neuritic extension of GFP(+) process. The neurons were transfected with AAV on 4 DIV and analyzed on 24 DIV. (a) An averaged image of multiple confocal planes across GFP(+) cells. (b) Sholl analysis of GFP(+) cells. Cells transfected with AAV-ctl presented neuritis with an increased ramification compared with cells transfected with AAV-hGAT1 and AAV-hGATA2 (*P<0.05). (c) Representative images are shown of high-magnification Z-stack projections of dendrites of GFP(+) cells from AAV-ctl- and AAV-hGATA-transfected cells. A continuous stretch of dendritic processes was imaged. (d) The spine density (arrowheads) of proximal (P) and distal (D) segments was significantly decreased in AAV-hGATA-transfected cells compared to AAV-ctl-transfected rats (*P<0.05, **P<0.01 compared to proximal region of AAV-ctl; #P<0.05, ###P<0.001 compared to distal region of AAV-ctl; n = 9–12 neurons in each group). The data were expressed as the number of spines per 10 µm. (e) MAP2, PSD-95, Tuj1 and GAP-43 expression in the cells described in (a) determined by Western blot analysis. Expression levels are depicted relative to the level of cells that were not exposed to virus (: no virus) for comparison. (n = 3–4 biological replicates per group). *P<0.05, **P<0.01, ***P<0.001 compared to control vector. Scale bars: a, 30 µm; c, 10 µm. Student's t-test.
Figure 3.
AAV-GATA infusions into hippocampus produce depression-like behavioral actions.
(a) Experimental design. Animals were injected with AAV-ctl, AAV-hGATA1 or AAV-hGATA2 and 5 weeks later were tested in behavioral paradigms, and then hippocampal sections were harvested for and immunohistochemistry (IHC) and biochemistry. (b) Representative images of GFP(+) cells in DG from AAV-ctl-eGFP, AAV-hGATA1-eGFP and AAV-hGATA2-eGFP injected animals. (c) Quantitative RT-PCR analysis was conducted to examine the expression of hGATA1 and hGATA-2 mRNA (**P<0.01). (d) FST. AAV-hGATA2 rats had a longer immobility score (time in seconds) than AAV-ctl-injected rats during the initial testing for 300 sec (F1,12 = 11.04, P = 0.005) and whole testing session for 900 sec (F1,12 = 5.12, P = 0.042). AAV-hGATA1 rats had a longer immobility score than AAV-ctl-injected rats during the initial testing for 300 sec (F1,12 = 5.79, P = 0.034; 900 sec: F1,12 = 0.52, P = 0.48). (e) Locomoter activity. The total distance moved in the box was similar between groups (F1,10 = 0.03, P = 0.84). (f) LH. AAV-hGATA1 or AAV-hGATA2 transduced rats had more escape failures than AAV-ctl animals (AAV-hGATA1: F1,20 = 7.09, P = 0.028; AAV-hGATA2: F1,19 = 5.85, P = 0.038). (g) SPT. Sucrose preference was not different both in AAV-hGATA1- or AAV-hGATA2-injected animals compared to AAV-ctl-injected animals (AAV-hGATA1: F1,19 = 0.08, P = 0.67; AAV-hGATA2: F1,19 = 0.19, P = 0.78). (h) NSFT. No differences in the latency to feed were shown between AAV-hGATA-injected animals and AAV-ctl-injected animals (AAV-hGATA1: F1,19 = 0.01, P = 0.92; AAV-hGATA2: F1,19 = 0.09, P = 0.76). (i-j) Home cage feeding and total fluid consumption. There was no difference in the home cage food intake (AAV-hGATA1: F1,19 = 0.08, P = 0.76; AAV-hGATA2: F1,19 = 0.79, P = 0.38) and total fluid consumption (AAV-hGATA1: F1,19 = 0.079, P = 0.78; AAV-hGATA2: F1,19 = 0.19, P = 0.66) between AAV-ctl-injected and AAV-hGATA-injected rats. Values represent mean ± s.e.m. from AAV-ctl (n = 10), AAV-hGATA1 (n = 9–11) and AAV-hGATA2 (n = 8–11). Student's t-test (c). ANOVA test (d-j). *P<0.05, **P<0.01 compared to control vector. Scale bar: 200 µm.
Figure 4.
Transcriptional repression of synapse-related genes and a decrease in spine density by AAV-hGATA1 and AAV-hGATA2 in DG of the rat hippocampus.
(a–d) Quantitative analysis of synapse-related genes in DG microdissected from rat hippocampus transfected with AAV-hGATA1 or AAV-hGATA2. Rab4b, AAV-hGATA1: F1,4 = 10.78, P = 0.03; AAV-hGATA2: F1,4 = 46.2, P = 0.002; PSD95, AAV-hGATA1: F1,4 = 31.73, P = 0.004; AAV-hGATA2: F1,4 = 42.8, P = 0.002; GAP43, AAV-hGATA1: F1,4 = 49.8, P = 0.002; AAV-hGATA2: F1,4 = 755.17, P = 0.00001; MAP2, AAV-hGATA1: F1,4 = 46.1, P = 0.002; AAV-hGATA2: F1,4 = 8.3, P = 0.04; vGlut1, AAV-hGATA1: F1,4 = 16.6, P = 0.015; AAV-hGATA2: F1,4 = 24.09, P = 0.02; GAD1, AAV-hGATA1: F1,4 = 0.004, P = 0.95; AAV-hGATA2: F1,4 = 1.59, P = 0.27. (g) Representative images are shown of high-magnification Z-stack projections of apical tuft segments of GFP(+) DG granule cells from AAV-ctl, AAV-hGATA1 and AAV-hGATA2-injected rats. (h) Density of dendritic spines (arrowheads) was significantly decreased in AAV-hGATA1- and AAV-hGATA2-injected rats compared with AAV-ctl-injected rats (a main effect of virus, F2,13 = 12.9, P<0.0001; AAV-hGATA1: F1,10 = 21.15, P = 0.0009; AAV-hGATA2: F1,7 = 5.63, P = 0.04). The data were expressed as the number of spines per 10 µm. ANOVA test. *P<0.05, **P<0.01, ***P<0.001 compared to control vector. Values represent mean ± s.e.m. of 5–7 cells from three animals per group. Scale bar: 10 µm.
Figure 5.
shGATA-2 increases synapse-related genes and spine density.
(a–b) Transfection with shGATA-2 caused ∼60% knockdown of GATA2 mRNA and protein expression in rat hippocampal cells. Western blotting analysis was conducted to examine the expression of endogeneous rat GATA-2, and representative blots were shown. The GATA-2 (51 kD) protein in the nuclear extracts is downregulated in response to transfection with shGATA-2 compared to control vector. (c–f) Quantitative analysis of synapse-related genes in rat hippocampal neurons transfected with shGATA-2. Rab4b, P<0.05; PSD95, P<0.01; GAP43, P<0.05; MAP2, P<0.05). (g) Representative images are shown of Z-stack projections of dendrites of MAP2(+) and GFP(+) cells from ctl- and shGATA2-transfected cells. A continuous stretch of dendritic processes was imaged. (right panel) high magnification. (h) Density of dendritic spines (arrowheads) was significantly increased in shGATA2-transfected cells compared with controls (***P<0.001 compared to controls). The data were expressed as the number of spines per 10 µm. n = 5–7 cells. (i) The number of dendritic branches exiting the soma (1′ dendrites), dendritic branches arborizing from 1′ dendrites (2′ dendrites, arrowheads) and dendritic branches arborizing from 2′ dendrites (3′ dendrites, arrows) were counted in neurons immunostained for MAP2 and GFP (n = 10–15 cells per treatment). Student's t test. *P<0.05, **P<0.01, ***P<0.001 compared to control vector. Values represent mean ± s.e.m. Scale bar: 10 µm.