Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods
Aim. Evidence-backed execution summary for Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods from Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Tissue preparation
reagent used in the protocol.
- Use
- Brain tissue for the TUNEL assay (see below) was rapidly frozen in liquid nitrogen and cut on a cryostat at 15 µm. Sections were mounted onto Superfrost coated glass slides, air-dried, post-fixed with 4% paraformaldehyde (20 min) and thoroughly washed in phosphate buffered saline (PBS).
Tissue preparation
reagent used in the protocol.
- Use
- The mice were killed with an overdose of ketamine and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed from the skull, were kept in the fixative for 24 h and then transferred into 30% sucrose, where they remained until equilibrium. Forty µm coronal sec...
Western blot
reagent used in the protocol.
- Use
- We confirmed the specificity of our antibody against doublecortin, on which the results of the present study are based, using a western blot of homogenate from mouse hippocampus (not shown). The antibody showed the expected band below 50 kD. This reaction was completely blocked with the DCX peptide.
Western blot
reagent used in the protocol.
- Use
- Mouse hippocampi were lysed with RIPA buffer (150 mM NaCl, 10% glycerol, 0.5 mM EDTA, 0.5% triton X-100, 1 mM phenylmethylsulphonyl fluoride, 25 µg/ml leupeptin, 25 µg/ml aprotinin and 1 mM sodium orthovanadate in 50 mM Tris-HCl, pH 7.6) and homogenized with an ultrasonic homogenizer (setting 40 Hz) for 60...
TUNEL assay
reagent used in the protocol.
- Use
- The TUNEL (terminal desoxy transferase-mediated dUTP nick end labeling) method visualizes apoptotic cells by detecting free 3' ends of DNA strand breaks [ ]. We used the ApopTag Fluorescein kit (Quantum Appligene). Brain sections were delipidated in ethanol/acetic acid (2:1) at -20°C. Afterwards the sections we...
Down-regulation of neurogenesis similarly does not affect dendritic maturation
We have recently shown that unlike in rats, training the Morris water maze led to a reduction in adult hippocampal neurogenesis in mice, presumably in response to the stress associated with swimming [ ], although this explanation remains speculative at present. Stress is an often described negative regulator of adul...
- Use
- We have recently shown that unlike in rats, training the Morris water maze led to a reduction in adult hippocampal neurogenesis in mice, presumably in response to the stress associated with swimming [ ], although this explanation remains speculative at present. Stress is an often described negative regulator of adul...
Immunohistochemistry
To determine the number of BrdU-labeled and DCX-expressing cells we used the peroxidase method (ABC system, Vectastain, Vector Laboratories) with biotinylated donkey anti-rat and anti-rabbit antibodies (1:500; Dianova) and nickel-intensified diaminobenzidine as chromogen. Immunofluorescent triple labeling was done a...
- Use
- To determine the number of BrdU-labeled and DCX-expressing cells we used the peroxidase method (ABC system, Vectastain, Vector Laboratories) with biotinylated donkey anti-rat and anti-rabbit antibodies (1:500; Dianova) and nickel-intensified diaminobenzidine as chromogen. Immunofluorescent triple labeling was done a...
Methods
For the water maze experiment, 21 female Nestin-GFP reporter mice [ ] (C57BL/6, 10 weeks of age at the beginning of the experiment) were used and randomly assigned to one of the following experimental groups: Morris water maze hidden version, Morris water maze cued version, standard laboratory conditions. On the 3 d...
- Use
- For the water maze experiment, 21 female Nestin-GFP reporter mice [ ] (C57BL/6, 10 weeks of age at the beginning of the experiment) were used and randomly assigned to one of the following experimental groups: Morris water maze hidden version, Morris water maze cued version, standard laboratory conditions. On the 3 d...
Western blot
Mouse hippocampi were lysed with RIPA buffer (150 mM NaCl, 10% glycerol, 0.5 mM EDTA, 0.5% triton X-100, 1 mM phenylmethylsulphonyl fluoride, 25 µg/ml leupeptin, 25 µg/ml aprotinin and 1 mM sodium orthovanadate in 50 mM Tris-HCl, pH 7.6) and homogenized with an ultrasonic homogenizer (setting 40 Hz) for 60...
- Use
- Mouse hippocampi were lysed with RIPA buffer (150 mM NaCl, 10% glycerol, 0.5 mM EDTA, 0.5% triton X-100, 1 mM phenylmethylsulphonyl fluoride, 25 µg/ml leupeptin, 25 µg/ml aprotinin and 1 mM sodium orthovanadate in 50 mM Tris-HCl, pH 7.6) and homogenized with an ultrasonic homogenizer (setting 40 Hz) for 60...
Analysis of newly generated cell phenotypes
One-in-twelve series of sections from animals of each group were triple-labeled for BrdU, GFP and DCX or double-labeled for BrdU and DCX. Fluorescent signals were detected using a spectral confocal microscope (Leica TCS SP2). All analyses were performed in sequential scanning mode to rule out cross-bleeding between...
- Use
- One-in-twelve series of sections from animals of each group were triple-labeled for BrdU, GFP and DCX or double-labeled for BrdU and DCX. Fluorescent signals were detected using a spectral confocal microscope (Leica TCS SP2). All analyses were performed in sequential scanning mode to rule out cross-bleeding between...
Analysis of newly generated cell phenotypes
From each animal and for every combination of phenotypes 50 BrdU-positive cells within the granule cell layer were analyzed at the confocal microscope for co-expression of the different markers. Relative numbers were related to the absolute counts of BrdU-positive cells per granule cell layer to yield the absolute n...
- Use
- From each animal and for every combination of phenotypes 50 BrdU-positive cells within the granule cell layer were analyzed at the confocal microscope for co-expression of the different markers. Relative numbers were related to the absolute counts of BrdU-positive cells per granule cell layer to yield the absolute n...
Figures and Tables
Distribution of dendritic morphologies among DCX-positive cells. A, among all DCX-positive cells of the SGZ, 22% are in categories A and B, the actively dividing stages (cf. Fig. 2). If categories C and D are included in this consideration the fraction of potentially proliferative DCX-positive cells is about one t...
- Use
- Distribution of dendritic morphologies among DCX-positive cells. A, among all DCX-positive cells of the SGZ, 22% are in categories A and B, the actively dividing stages (cf. Fig. 2). If categories C and D are included in this consideration the fraction of potentially proliferative DCX-positive cells is about one t...
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The duration of DCX expression varies in the course of neuronal development
To assess the progression through the developmental stages, we used a time course study, in which BrdU was injected once and the brains were examined at different time points after the injection. The overall pattern was consistent with development in the sense of a progression from less mature to more mature appearing stages. At early time points after BrdU, DCX-positive cells were mainly found in categories A through C (Fig. ); at 4 weeks after BrdU, the majority of cells had reached categories E and F. However, at late time points after BrdU (7 days and 4 weeks) a considerable number of DCX-positive cells was found in categories A through D, indicating either asymmetric divisions on these stages or strongly varying dynamics of development. As early as 1 day after BrdU the first cells were seen in postmitotic category E (but not F). This is consistent with previous observations that...
The duration of DCX expression varies in the course of neuronal development
At 1 and 3 days after BrdU, we examined the distribution of BrdU/DCX/CR-triple-positive cells into the morphological classes A through F (Fig. ). We found a shift towards more mature stages between 1 and 3 days. The absence of any BrdU/DCX/CR-triple-positive cells in category F at 3 days after BrdU told us that it takes more than 3 days after exit from the cell cycle to reach this level of maturity. But more importantly, we found BrdU/DCX/CR-triple-positive cells in the most immature stages. This implies that postmitotic cells are found in categories A, B, and C, which can otherwise (if CR negative) also be associated with proliferative activity. Cells might leave the cell cycle, while DCX-positive and express CR at varying time-points (here represented by the different categories) thereafter.
Tissue preparation
Brain tissue for the TUNEL assay (see below) was rapidly frozen in liquid nitrogen and cut on a cryostat at 15 µm. Sections were mounted onto Superfrost coated glass slides, air-dried, post-fixed with 4% paraformaldehyde (20 min) and thoroughly washed in phosphate buffered saline (PBS).
Dynamic of neuronal maturation is not influenced by neurogenic stimuli
When the DCX-positive cells were categorized into the classes A to F, we found that the distribution did not differ from controls. Although the absolute numbers in each category increased (significantly except for classes A and F), the distribution in the different categories was not influenced. We interpret this result as indicating that at one week after the neurogenic stimulus, progression of DCX-positive cells through the developmental stages highlighted by DCX expression was constant. The number of cells had increased but not their (variable) rate of further development. This might imply that the variability in the duration of DCX expression is not affected by the microenvironmental condition prevalent after experimental seizures. Whereas one study showed an acute proliferative response of GFAP-positive radial cells after experimental seizures [ ], one of our own recent studies f...
Methods
The experiments were largely done on material from a previously published study [ ]. A set of 6 week-old 24 female C57BL/6 mice (19-24 g; Charles River) were divided into 6 groups and perfused 4 hours (N = 5), 1 day (N = 5), 3 days (N = 5), 1 week (N = 5), 2.5 weeks (N = 4) and 4 weeks (N = 5) after a single intraperitoneal BrdU-injection (5-Bromo-2-deoxyuridine, 50 mg/kg BrdU in sterile 0,9% NaCl; Sigma).
Methods
Additional 18 female C57BL/6 mice (same age as above) were divided into 3 groups: seizure (N = 6), running (N = 6) and controls (N = 6). Seizure animals received a single intraperitoneal application of 30 mg/kg kainic acid (KA, Sigma) in 0.1 M phosphate buffered saline (PBS) on the day before BrdU-injections (Day 0), and only those displaying continuous convulsive seizure activity were used in these experiments. The "Runner" group was housed with 2-3 animals per cage that was equipped with a running wheel. During the first 7 days of the experiment all animals received one daily injection of BrdU. Tissue from this experiment will be used in an unrelated study. Data on dendritic morphology, etc., were exclusively generated for the present study.
Methods
To analyze the spatial relationship between DCX-positive cells and astrocytes we used 3 female transgenic mice expressing enhanced green fluorescent protein EGFP under the promoter for glial fibrillary acidic protein (GFAP). The animals were kindly provided by Helmut Kettenmann, Berlin [ ] and were 7 weeks of age. For the detection of apoptosis a total of 37 female C57BL/6J mice (Charles River, Sulzfeld, Germany), 8 weeks of age, were used.
Methods
For the water maze experiment, 21 female Nestin-GFP reporter mice [ ] (C57BL/6, 10 weeks of age at the beginning of the experiment) were used and randomly assigned to one of the following experimental groups: Morris water maze hidden version, Morris water maze cued version, standard laboratory conditions. On the 3 days before the experiment each animal received single injection of BrdU. Animals were subjected to water maze training at days 5 to 8 of the experiment (days 5 to 8 after the last BrdU injection). We followed the protocol devised by Wolfer and Lipp [ ]. Six trials of training each maximally lasting for 2 minutes were given each day. Tissue from that experiment that has been published elsewhere [ ] was analyzed for the present study. Again, the data on dendritic morphology, etc., were exclusively generated for the present study.
Measurement outputs
What raw and processed outputs should exist?
We did not find a clear preferential association of the most immature categories with the astrocytes although more cells with astrocytic contact were found in categories A ̵...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Seki and Arai were the first to propose a tight association of developing new neurons and radial glia elements [ ]. Seri et al. reported a close spatial relationship between DCX...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
We asked how the close association between maturing neurons and astrocyte-like cells would relate to the stages of maturation. To visualize GFAP-positive cells on a light micros...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
DCX-positive cells either did not show any contact to GFAP-positive cells (6.8%), were touched by an astrocytic process (15.8%), or were surrounded by GFAP-positive processes li...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
As described previously, in the case of wheel running we did not find a significant increase in the number of DCX-positive cells at this time-point but an increase in cell proliferation [ ].
from paperScoring or quantification
Quantify the primary readouts for this experiment: We did not find a clear preferential association of the most immature categories with the astrocytes although more cells with astrocytic contact were found in categories A ̵...; Seki and Arai were the first to propose a tight association of developing new neurons and radial glia elements [ ]. Seri et al. reported a close spatial relationship between DCX...; We asked how the close association between maturing neurons and astrocyte-like cells would relate to the stages of maturation. To visualize GFAP-positive cells on a light micros...; DCX-positive cells either did not show any contact to GFAP-positive cells (6.8%), were touched by an astrocytic process (15.8%), or were surrounded by GFAP-positive processes li....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
As described previously, in the case of wheel running we did not find a significant increase in the number of DCX-positive cells at this time-point but an increase in cell proli...; All numerical analyses were performed using Statview 5.0.1. For all comparisons ANOVA was performed followed by Fisher's post hoc test, if appropriate. Differences were consider...
from paperReporting output
Report representative outputs alongside summary comparisons for We did not find a clear preferential association of the most immature categories with the astrocytes although more cells with astrocytic contact were found in categories A ̵..., Seki and Arai were the first to propose a tight association of developing new neurons and radial glia elements [ ]. Seri et al. reported a close spatial relationship between DCX..., We asked how the close association between maturing neurons and astrocyte-like cells would relate to the stages of maturation. To visualize GFAP-positive cells on a light micros..., DCX-positive cells either did not show any contact to GFAP-positive cells (6.8%), were touched by an astrocytic process (15.8%), or were surrounded by GFAP-positive processes li....
inferred from protocolStructured statistical methods
As described previously, in the case of wheel running we did not find a significant increase in the number of DCX-positive cells at this time-point but an increase in cell proli...; All numerical analyses were performed using Statview 5.0.1. For all comparisons ANOVA was performed followed by Fisher's post hoc test, if appropriate. Differences were consider...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
To assess the progression through the developmental stages, we used a time course study, in which BrdU was injected once and the brains were examined at different time points after the injection. The overall pattern was consistent with development in the sense of a progression from less mature to more mature appearing stages. At early time points after BrdU, DCX-positive cells were mainly found in categories A through C (Fig. ); at 4 weeks after BrdU, the majority of cells had reached categories E and F. However, at late time points after BrdU (7 days and 4 weeks) a considerable number of DCX-positive cells was found in categories A through D, indicating either asymmetric divisions on these stages or strongly varying dynamics of development. As early as 1 day after BrdU the first cells were seen in postmitotic category E (but not F). This is consistent with previous observations that new neurons can show very rapid signs of postmitotic maturation after the last cell division [, ] and the presence of BrdU/DCX/CR-triple-positive cells at 1 day after BrdU (Fig. ). Consequently, the total duration of the phase of DCX expression appears to vary. Even at 4 weeks after BrdU the number...
At 1 and 3 days after BrdU, we examined the distribution of BrdU/DCX/CR-triple-positive cells into the morphological classes A through F (Fig. ). We found a shift towards more mature stages between 1 and 3 days. The absence of any BrdU/DCX/CR-triple-positive cells in category F at 3 days after BrdU told us that it takes more than 3 days after exit from the cell cycle to reach this level of maturity. But more importantly, we found BrdU/DCX/CR-triple-positive cells in the most immature stages. This implies that postmitotic cells are found in categories A, B, and C, which can otherwise (if CR negative) also be associated with proliferative activity. Cells might leave the cell cycle, while DCX-positive and express CR at varying time-points (here represented by the different categories) thereafter.
Brain tissue for the TUNEL assay (see below) was rapidly frozen in liquid nitrogen and cut on a cryostat at 15 µm. Sections were mounted onto Superfrost coated glass slides, air-dried, post-fixed with 4% paraformaldehyde (20 min) and thoroughly washed in phosphate buffered saline (PBS).
When the DCX-positive cells were categorized into the classes A to F, we found that the distribution did not differ from controls. Although the absolute numbers in each category increased (significantly except for classes A and F), the distribution in the different categories was not influenced. We interpret this result as indicating that at one week after the neurogenic stimulus, progression of DCX-positive cells through the developmental stages highlighted by DCX expression was constant. The number of cells had increased but not their (variable) rate of further development. This might imply that the variability in the duration of DCX expression is not affected by the microenvironmental condition prevalent after experimental seizures. Whereas one study showed an acute proliferative response of GFAP-positive radial cells after experimental seizures [ ], one of our own recent studies found that at the peak of proliferation after kainic acid-induced seizures the increased proliferative activity can be primarily attributed to DCX-expressing type-3 cells, many of which then show an ectopic position in the granule cell layer [ ] (see also Fig. ). The present data add to this finding...
The experiments were largely done on material from a previously published study [ ]. A set of 6 week-old 24 female C57BL/6 mice (19-24 g; Charles River) were divided into 6 groups and perfused 4 hours (N = 5), 1 day (N = 5), 3 days (N = 5), 1 week (N = 5), 2.5 weeks (N = 4) and 4 weeks (N = 5) after a single intraperitoneal BrdU-injection (5-Bromo-2-deoxyuridine, 50 mg/kg BrdU in sterile 0,9% NaCl; Sigma).
Additional 18 female C57BL/6 mice (same age as above) were divided into 3 groups: seizure (N = 6), running (N = 6) and controls (N = 6). Seizure animals received a single intraperitoneal application of 30 mg/kg kainic acid (KA, Sigma) in 0.1 M phosphate buffered saline (PBS) on the day before BrdU-injections (Day 0), and only those displaying continuous convulsive seizure activity were used in these experiments. The "Runner" group was housed with 2-3 animals per cage that was equipped with a running wheel. During the first 7 days of the experiment all animals received one daily injection of BrdU. Tissue from this experiment will be used in an unrelated study. Data on dendritic morphology, etc., were exclusively generated for the present study.
To analyze the spatial relationship between DCX-positive cells and astrocytes we used 3 female transgenic mice expressing enhanced green fluorescent protein EGFP under the promoter for glial fibrillary acidic protein (GFAP). The animals were kindly provided by Helmut Kettenmann, Berlin [ ] and were 7 weeks of age. For the detection of apoptosis a total of 37 female C57BL/6J mice (Charles River, Sulzfeld, Germany), 8 weeks of age, were used.
For the water maze experiment, 21 female Nestin-GFP reporter mice [ ] (C57BL/6, 10 weeks of age at the beginning of the experiment) were used and randomly assigned to one of the following experimental groups: Morris water maze hidden version, Morris water maze cued version, standard laboratory conditions. On the 3 days before the experiment each animal received single injection of BrdU. Animals were subjected to water maze training at days 5 to 8 of the experiment (days 5 to 8 after the last BrdU injection). We followed the protocol devised by Wolfer and Lipp [ ]. Six trials of training each maximally lasting for 2 minutes were given each day. Tissue from that experiment that has been published elsewhere [ ] was analyzed for the present study. Again, the data on dendritic morphology, etc., were exclusively generated for the present study.
Machine-readable layer
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"name": "Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods",
"description": "Evidence-backed execution summary for Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods from Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation.",
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"text": "To assess the progression through the developmental stages, we used a time course study, in which BrdU was injected once and the brains were examined at different time points after the injection. The overall pattern was consistent with development in the sense of a progression from less mature to more mature appearing stages. At early time points after BrdU, DCX-positive cells were mainly found in categories A through C (Fig. ); at 4 weeks after BrdU, the majority of cells had reached categories E and F. However, at late time points after BrdU (7 days and 4 weeks) a considerable number of DCX-positive cells was found in categories A through D, indicating either asymmetric divisions on these stages or strongly varying dynamics of development. As early as 1 day after BrdU the first cells were seen in postmitotic category E (but not F). This is consistent with previous observations that..."
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"text": "When the DCX-positive cells were categorized into the classes A to F, we found that the distribution did not differ from controls. Although the absolute numbers in each category increased (significantly except for classes A and F), the distribution in the different categories was not influenced. We interpret this result as indicating that at one week after the neurogenic stimulus, progression of DCX-positive cells through the developmental stages highlighted by DCX expression was constant. The number of cells had increased but not their (variable) rate of further development. This might imply that the variability in the duration of DCX expression is not affected by the microenvironmental condition prevalent after experimental seizures. Whereas one study showed an acute proliferative response of GFAP-positive radial cells after experimental seizures [ ], one of our own recent studies f..."
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"text": "The experiments were largely done on material from a previously published study [ ]. A set of 6 week-old 24 female C57BL/6 mice (19-24 g; Charles River) were divided into 6 groups and perfused 4 hours (N = 5), 1 day (N = 5), 3 days (N = 5), 1 week (N = 5), 2.5 weeks (N = 4) and 4 weeks (N = 5) after a single intraperitoneal BrdU-injection (5-Bromo-2-deoxyuridine, 50 mg/kg BrdU in sterile 0,9% NaCl; Sigma)."
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"text": "Additional 18 female C57BL/6 mice (same age as above) were divided into 3 groups: seizure (N = 6), running (N = 6) and controls (N = 6). Seizure animals received a single intraperitoneal application of 30 mg/kg kainic acid (KA, Sigma) in 0.1 M phosphate buffered saline (PBS) on the day before BrdU-injections (Day 0), and only those displaying continuous convulsive seizure activity were used in these experiments. The \"Runner\" group was housed with 2-3 animals per cage that was equipped with a running wheel. During the first 7 days of the experiment all animals received one daily injection of BrdU. Tissue from this experiment will be used in an unrelated study. Data on dendritic morphology, etc., were exclusively generated for the present study."
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"text": "To analyze the spatial relationship between DCX-positive cells and astrocytes we used 3 female transgenic mice expressing enhanced green fluorescent protein EGFP under the promoter for glial fibrillary acidic protein (GFAP). The animals were kindly provided by Helmut Kettenmann, Berlin [ ] and were 7 weeks of age. For the detection of apoptosis a total of 37 female C57BL/6J mice (Charles River, Sulzfeld, Germany), 8 weeks of age, were used."
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"name": "Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods",
"item": "https://replicatescience.com/experiments/variability-of-doublecortin-associated-dendrite-maturation-in-adult-hippocampal-neurogenesis-is-independent-of-the-regulation-of-precursor-cell-proliferation-methods-tobias-pl-252-/variability-of-doublecortin-associated-dendrite-maturation-in-adult-hippocampal-neurogenesis-is-inde-mlph87xh"
}
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]