Journal: Scientific Reports

Article Title: EAAC1 gene deletion reduces adult hippocampal neurogenesis after transient cerebral ischemia

doi: 10.1038/s41598-018-25191-4

Figure Lengend Snippet: EAAC1 is expressed in mature and immature neurons. ( A ) Representative immunofluorescence images show DCX (green) and EAAC1 (red) immunoreactive (DCX+/ EAAC1 +) cells in the DG. ( B ) Representative immunofluorescence images reveal Tuj1 (green) and EAAC1 (red) immunoreactive (Tuj1+/ EAAC1 +) cells in the DG. ( C ) Representative immunofluorescence images show MAP2 (green) and EAAC1 (red) immunoreactive (MAP2+/ EAAC1 +) cells in the DG and CA1. Scale bar = 10 μm.

Article Snippet: We used the following antibodies as primary antibodies; guinea pig polyclonal anti-DCX (diluted 1:1k, Millipore), mouse monoclonal anti-beta 3 tubulin (TUJ1, diluted 1:500, Abcam), mouse monoclonal anti-microtubule-associated protein 2 (MAP2, for recognizing a neuron-specific cytoskeletal protein, diluted 1:200, Millipore), and rabbit polyclonal anti-excitatory amino acid transporter 3 ( EAAT3 , also known as EAAC1 , diluted 1:200, Almone labs, Jerusalem, Israel).

Techniques: Immunofluorescence

Journal: Scientific Reports

Article Title: EAAC1 gene deletion reduces adult hippocampal neurogenesis after transient cerebral ischemia

doi: 10.1038/s41598-018-25191-4

Figure Lengend Snippet: Ischemia-induced progenitor cell proliferation and neuroblast production are reduced in EAAC1 −/− mice. ( A ) The schematic diagram shows the experimental procedure used in this study. BrdU was intraperitoneally injected twice per day for 4 consecutive days from day 3 after ischemia. Mice were killed on day 7. ( B , C , F ) Representative images reveal BrdU- ( B ), Ki67- ( C ) and DCX-labeled cells ( F ) in the hippocampal dentate gyrus (DG) from young and aged mice (either WT or EAAC1 −/− ) at 7 days after ischemia. BrdU- and Ki67-labeled cells are located in the subgranular zone (SGZ) of the DG and appear most often in clusters. Detection of BrdU and Ki67 is accomplished with DAB. Ki67-expressing cells are indicated with an arrow. DCX-labeled cells are also located in the inner granular layer. Scale bar = 100 μm. (D,E G) Bar graph shows the number of BrdU- ( D ), Ki67- ( E ) and DCX-labeled cells ( G ) in the SGZ from young and aged mice (WT or EAAC1 −/− ) at 7 days after ischemia. Data are expressed as the mean + SEM; n = 7–9 from each group, # p < 0.05 versus young mice.

Article Snippet: We used the following antibodies as primary antibodies; guinea pig polyclonal anti-DCX (diluted 1:1k, Millipore), mouse monoclonal anti-beta 3 tubulin (TUJ1, diluted 1:500, Abcam), mouse monoclonal anti-microtubule-associated protein 2 (MAP2, for recognizing a neuron-specific cytoskeletal protein, diluted 1:200, Millipore), and rabbit polyclonal anti-excitatory amino acid transporter 3 ( EAAT3 , also known as EAAC1 , diluted 1:200, Almone labs, Jerusalem, Israel).

Techniques: Injection, Labeling, Expressing

Journal: Scientific Reports

Article Title: EAAC1 gene deletion reduces adult hippocampal neurogenesis after transient cerebral ischemia

doi: 10.1038/s41598-018-25191-4

Figure Lengend Snippet: Genetic deletion of EAAC1 reduces survival of newly generated neurons after ischemia. ( A ) The schematic diagram shows the experimental procedure used in this study. BrdU was intraperitoneally injected twice per day for 4 consecutive days from day 3 after ischemia. Mice were killed on day 30. ( B ) Photomicrographs show the distribution of BrdU-labeled cells in the SGZ/GCL of the hippocampus of young and aged mice (WT or EAAC1 −/− ) after ischemia. Newborn cells were labeled with BrdU, and their survival was assessed 30 days after ischemia. Scale bar = 100 μm. ( C ) Graph shows the number of BrdU-labeled cells in the SGZ/GCL of young and aged mice (WT or EAAC1 −/− ) at 30 days after ischemia. Data are expressed by the mean + SEM; n = 3–7 from each group, * p < 0.05 versus WT mice; # p < 0.05 versus young mice. (D,E) Representative immunofluorescence images show the phenotype of cells that have survived in the SGZ/GCL. Scale bar = 20 μm. ( F , G ) Bar graph represents the number of BrdU-labeled cells that were colocalized with NeuN (F) or GFAP (G) in the SGZ/GCL from young and aged mice (WT or EAAC1 −/− ) at 30 days after ischemia. Data are expressed as the mean + SEM; n = 3–7 from each group, * p < 0.05 versus WT mice; # p < 0.05 versus young mice.

Article Snippet: We used the following antibodies as primary antibodies; guinea pig polyclonal anti-DCX (diluted 1:1k, Millipore), mouse monoclonal anti-beta 3 tubulin (TUJ1, diluted 1:500, Abcam), mouse monoclonal anti-microtubule-associated protein 2 (MAP2, for recognizing a neuron-specific cytoskeletal protein, diluted 1:200, Millipore), and rabbit polyclonal anti-excitatory amino acid transporter 3 ( EAAT3 , also known as EAAC1 , diluted 1:200, Almone labs, Jerusalem, Israel).

Techniques: Generated, Injection, Labeling, Immunofluorescence

Journal: Scientific Reports

Article Title: EAAC1 gene deletion reduces adult hippocampal neurogenesis after transient cerebral ischemia

doi: 10.1038/s41598-018-25191-4

Figure Lengend Snippet: Progenitor cell proliferation and neuroblast production are not different between WT and EAAC1 −/− mice. ( A ) The schematic diagram shows the experimental procedure used in this study. BrdU was intraperitoneally injected twice per day for 4 consecutive days from day 3 after sham surgery. Mice were killed on day 7. ( B , C , F ) Representative images reveal BrdU- ( B ) Ki67- ( C ) and DCX-labeled cells ( F ) in the hippocampal DG of young and aged mice (WT or EAAC1 −/− ) at 7 days after sham surgery. The Ki67-expressing cells are indicated by an arrow. Scale bar = 100 μm. (D,E,G) The bar graph shows the number of BrdU- ( D ) Ki67- ( E ), and DCX-labeled cells ( G ) in the SGZ from young and aged mice (WT or EAAC1 −/− ) at 7 days after sham surgery. Data are expressed as the mean + SEM; n = 6–10 from each group, # p < 0.05 versus young mice.

Article Snippet: We used the following antibodies as primary antibodies; guinea pig polyclonal anti-DCX (diluted 1:1k, Millipore), mouse monoclonal anti-beta 3 tubulin (TUJ1, diluted 1:500, Abcam), mouse monoclonal anti-microtubule-associated protein 2 (MAP2, for recognizing a neuron-specific cytoskeletal protein, diluted 1:200, Millipore), and rabbit polyclonal anti-excitatory amino acid transporter 3 ( EAAT3 , also known as EAAC1 , diluted 1:200, Almone labs, Jerusalem, Israel).

Techniques: Injection, Labeling, Expressing

Journal: Scientific Reports

Article Title: EAAC1 gene deletion reduces adult hippocampal neurogenesis after transient cerebral ischemia

doi: 10.1038/s41598-018-25191-4

Figure Lengend Snippet: Genetic deletion of EAAC1 reduces survival of newborn neurons. ( A ) The schematic diagram shows the experimental procedure used in this study. BrdU was intraperitoneally injected twice per day for 4 consecutive days from day 3 after sham surgery. Mice were killed on day 30. ( B ) Photomicrographs show the distribution of BrdU-labeled cells in the SGZ/GCL of the hippocampus of young and aged mice (WT or EAAC1 −/− ). Newborn cells were labeled with BrdU, and their survival was assessed 30 days after sham surgery. Scale bar = 100 μm. ( C ) Graph shows the number of BrdU-labeled cells in the SGZ/GCL from young or aged mice (either WT or EAAC1 −/− ) at 30 days after sham surgery. Data are expressed as the mean + SEM; n = 6–9 from each group, * p < 0.05 versus WT mice; # p < 0.05 versus young mice. ( D , E ) The bar graph represents the number of BrdU-labeled cells that were colocalized with NeuN ( D ), or GFAP ( E ) in the SGZ/GCL of young and aged mice (WT or EAAC1 −/− ) at 30 days after sham surgery. Data are represented as the mean + SEM; n = 5–8 from each group, * p < 0.05 versus WT mice; # p < 0.05 versus young mice.

Article Snippet: We used the following antibodies as primary antibodies; guinea pig polyclonal anti-DCX (diluted 1:1k, Millipore), mouse monoclonal anti-beta 3 tubulin (TUJ1, diluted 1:500, Abcam), mouse monoclonal anti-microtubule-associated protein 2 (MAP2, for recognizing a neuron-specific cytoskeletal protein, diluted 1:200, Millipore), and rabbit polyclonal anti-excitatory amino acid transporter 3 ( EAAT3 , also known as EAAC1 , diluted 1:200, Almone labs, Jerusalem, Israel).

Techniques: Injection, Labeling

Journal: Scientific Reports

Article Title: EAAC1 gene deletion reduces adult hippocampal neurogenesis after transient cerebral ischemia

doi: 10.1038/s41598-018-25191-4

Figure Lengend Snippet: Proposed mechanism by which EAAC1 knockout reduces the survival of newly generated neurons. This schematic drawing indicates several chain reactions that are thought to occur after impairment of neuronal cysteine transport in EAAC1 −/− mice. ( A ) EAAC1 -mediated cysteine transport is essential for GSH synthesis in neurons. ( B ) Reduced cysteine uptake by EAAC1 knockout can result in reduced GSH synthesis. ( C ) Genetic deletion of EAAC1 attenuates neuronal antioxidant capacity by reduced GSH synthesis. This can create enhanced oxidative stress which subsequently has a negative impact on the survival of newly generated neurons.

Article Snippet: We used the following antibodies as primary antibodies; guinea pig polyclonal anti-DCX (diluted 1:1k, Millipore), mouse monoclonal anti-beta 3 tubulin (TUJ1, diluted 1:500, Abcam), mouse monoclonal anti-microtubule-associated protein 2 (MAP2, for recognizing a neuron-specific cytoskeletal protein, diluted 1:200, Millipore), and rabbit polyclonal anti-excitatory amino acid transporter 3 ( EAAT3 , also known as EAAC1 , diluted 1:200, Almone labs, Jerusalem, Israel).

Techniques: Knock-Out, Generated