Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
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?
Microscopy-based tissue or cell-state imaging
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Microscopy-based tissue or cell-state imaging.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Microscopy-based tissue or cell-state imaging.
inferred from protocolSource 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
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods",
"description": "Protocol for Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "The duration of DCX expression varies in the course of neuronal development",
"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..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "The duration of DCX expression varies in the course of neuronal development",
"text": "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."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Tissue preparation",
"text": "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)."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Dynamic of neuronal maturation is not influenced by neurogenic stimuli",
"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..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Methods",
"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)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Methods",
"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."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Methods",
"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."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Methods",
"text": "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."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation",
"datePublished": "2006",
"author": [
{
"@type": "Person",
"name": "Tobias Plümpe"
},
{
"@type": "Person",
"name": "Dan Ehninger"
},
{
"@type": "Person",
"name": "Barbara Steiner"
},
{
"@type": "Person",
"name": "Friederike Klempin"
},
{
"@type": "Person",
"name": "Sebastian Jessberger"
},
{
"@type": "Person",
"name": "Moritz Brandt"
},
{
"@type": "Person",
"name": "Benedikt Römer"
},
{
"@type": "Person",
"name": "Gerardo Ramirez Rodriguez"
},
{
"@type": "Person",
"name": "Golo Kronenberg"
},
{
"@type": "Person",
"name": "Gerd Kempermann"
}
],
"identifier": "10.1186/1471-2202-7-77"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"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"
}
]
}
]