Antidepressants recruit new neurons to improve stress response regulation methods
Aim. Evidence-backed execution summary for Antidepressants recruit new neurons to improve stress response regulation methods from Antidepressants recruit new neurons to improve stress response regulation.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Immunohistochemistry
reagent used in the protocol.
- Use
- Immunohistochemistry for subgranular zone (SGZ) proliferation was performed as previously described. Rat anti-BrdU antibody (1:500, Oxford Biotechnology, Oxfordshire, UK) was used as primary antibody followed by rabbit anti-rat biotinylated antibody (1:200, Vector Laboratories, Burlingame, CA, USA). To label Fos, im...
Immunohistochemistry
reagent used in the protocol.
- Use
- For immunohistochemistry based on fluorescence labeling, the different primary antibodies used were as follows: mouse anti-NeuN monoclonal antibody (1:1000, Chemicon International, Billerica, MA, USA), rat anti-BrdU monoclonal antibody (1:500 Oxford Biotechnology) and a rabbit anti-Fos monoclonal antibody (1:1000 Ox...
Quantification of corticosterone levels
reagent used in the protocol.
- Use
- Fecal samples were analyzed for immunoreactive corticosterone metabolites using a 5α-pregnane-3β,11β,21-triol-20-one enzyme-immunoassay as previously described. Details regarding development, biochemical characteristics and physiological validation of this assay have previously been described., Plasm...
Cookie test
This test required a device containing three aligned compartments with the same dimension (20 × 20 × 20 cm). Only the colors of the walls and the floor were different between the compartments (Figure 2a). Mice were first familiarized with a chocolate cookie (Pepito, Lu, France) 4.5 weeks before the f...
- Use
- This test required a device containing three aligned compartments with the same dimension (20 × 20 × 20 cm). Only the colors of the walls and the floor were different between the compartments (Figure 2a). Mice were first familiarized with a chocolate cookie (Pepito, Lu, France) 4.5 weeks before the f...
Image analysis and cell quantification
When staining was visualized with DAB, the number of positive cells was counted using × 20 and × 40 objective lens with a conventional light microscope. For Fos-labeled cell counting of subcortical regions, the nomenclature and nuclei boundaries used were those defined by Paxinos and Franklin's mouse brain...
- Use
- When staining was visualized with DAB, the number of positive cells was counted using × 20 and × 40 objective lens with a conventional light microscope. For Fos-labeled cell counting of subcortical regions, the nomenclature and nuclei boundaries used were those defined by Paxinos and Franklin's mouse brain...
Image analysis and cell quantification
When staining was visualized with fluorochrome-labeled secondary antibodies, the number of positive cells was counted using a × 40 objective lens with a confocal or an epifluorescence microscope. Eight sections (16 hippocampus) along the rostro-caudal extent of the hippocampus were examined; ∼bregma: W...
- Use
- When staining was visualized with fluorochrome-labeled secondary antibodies, the number of positive cells was counted using a × 40 objective lens with a confocal or an epifluorescence microscope. Eight sections (16 hippocampus) along the rostro-caudal extent of the hippocampus were examined; ∼bregma: W...
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UCMS
The stress regimen used was previously described and is a variant of the chronic mild stress procedures described by Willner in rats. UCMS mice were repeatedly subjected to various socio-environmental stressors according to an unpredictable schedule for a total period of 6-8 weeks. From the third week of UCMS onward, mice were administered intraperitoneal (ip), once a day, with either vehicle, fluoxetine (20 mg kg -1 per day) or SSR125543 (20 mg kg -1 per day). Treatment was always maintained until the end of the experiments. Body weight and coat state were assessed weekly, as markers of the progression of the UCMS-evoked syndrome.,
Cookie test
This test required a device containing three aligned compartments with the same dimension (20 × 20 × 20 cm). Only the colors of the walls and the floor were different between the compartments (Figure 2a). Mice were first familiarized with a chocolate cookie (Pepito, Lu, France) 4.5 weeks before the first testing. One hour before testing, the regular food was removed from the cage lid. At the time of testing, a small amount (2±1 g) of chocolate cookie (or regular food in a control experiment) was placed at the center of the black compartment. The mouse was initially placed in the white compartment of the apparatus. Each session of the test lasted five minutes. The cookie consumption (# bites) was recorded within the 5 min test period. Four sessions of testing were performed within 10 days (inter-test interval: 2 days).
Dexamethasone (DEX) administration procedures
DEX suppression test: mice were i.p. injected with either DEX-P (0.1 mg kg -1, five mice per group) or saline (0.9% NaCl, five mice per group). All fecalthe boli that were voided between 8 and 10 h after the injection were collected to measure the level of fecal corticosterone metabolites.
Dexamethasone (DEX) administration procedures
Intrahippocampal DEX infusion: the infusion procedure was derived from the method previously described. Mice were stereotaxically and bilaterally implanted with guide cannulas (6 mm long, 0.6 mm outer diameter, 0.36 mm inner diameter). The following coordinates were used for the guide cannula implantation: bregma=-3.08, lateral=±2.3, vertical=-1.4. After 1-week recovery period from the surgery, infusion cannulas (7 mm long, 0.3 mm outer diameter, 0.17 mm inner diameter) were placed and extended 1 mm below the guide cannulas. The animals received bilateral infusions during 1.5 min of either vehicle (0.9% NaCl/0.2% ethanol) or DEX (50 ng in 0.6 µl). In one experiment, the brain was collected 2 h following the infusion to assess DEX-induced Fos changes in downstream brain structures and the PVN (Figur...
Chronic stress disrupts the sensitivity of the granule cell network to novelty/glucocorticoid effects
We then directly tested the ability of the DG granule neurons to react to glucocorticoids. These cells can be activated and express immediate early genes when individuals are exposed to a new environment.,, We took advantage of this property to examine the sensitivity of new and older granule cells to a new environment exposure following a single injection of DEX-P or vehicle ( ). For this purpose, we concurrently labeled cells for the neuronal marker NeuN, the immediate early gene product Fos ('activated' granule cells) and the proliferation marker BrdU (4 week-old newborn cells) ( ).
Measurement outputs
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Our previous results raise the possibility that stimulating neurogenesis by fluoxetine may facilitate the regulation of stress systems. We therefore assessed whether UCMS and fl...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
When staining was visualized with DAB, the number of positive cells was counted using × 20 and × 40 objective lens with a conventional light microscope. For Fos-labele...
- 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
When staining was visualized with fluorochrome-labeled secondary antibodies, the number of positive cells was counted using a × 40 objective lens with a confocal or an epif...
- 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
Considering that relatively small sample sizes were used and that assumptions for parametric statistics could not be ensured (normality and homoscedasticity), data were analyzed...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Our previous results raise the possibility that stimulating neurogenesis by fluoxetine may facilitate the regulation of stress systems.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Our previous results raise the possibility that stimulating neurogenesis by fluoxetine may facilitate the regulation of stress systems. We therefore assessed whether UCMS and fl...; When staining was visualized with DAB, the number of positive cells was counted using × 20 and × 40 objective lens with a conventional light microscope. For Fos-labele...; When staining was visualized with fluorochrome-labeled secondary antibodies, the number of positive cells was counted using a × 40 objective lens with a confocal or an epif...; Considering that relatively small sample sizes were used and that assumptions for parametric statistics could not be ensured (normality and homoscedasticity), data were analyzed....
from paperStatistical comparison
Our previous results raise the possibility that stimulating neurogenesis by fluoxetine may facilitate the regulation of stress systems. We therefore assessed whether UCMS and fl...; To examine more deeply the link between hippocampal neurogenesis and stress response, we investigated whether ablation of neurogenesis and UCMS induce abnormalities in the HPA a...; Considering that relatively small sample sizes were used and that assumptions for parametric statistics could not be ensured (normality and homoscedasticity), data were analyzed...; We first assessed the effects on the whole granule cell population (, P =0.0209). Although UCMS-vehicle mice exhibited an apparent lower NeuN+/Fos+ cell density than the other...
from paperReporting output
Report representative outputs alongside summary comparisons for Our previous results raise the possibility that stimulating neurogenesis by fluoxetine may facilitate the regulation of stress systems. We therefore assessed whether UCMS and fl..., When staining was visualized with DAB, the number of positive cells was counted using × 20 and × 40 objective lens with a conventional light microscope. For Fos-labele..., When staining was visualized with fluorochrome-labeled secondary antibodies, the number of positive cells was counted using a × 40 objective lens with a confocal or an epif..., Considering that relatively small sample sizes were used and that assumptions for parametric statistics could not be ensured (normality and homoscedasticity), data were analyzed....
inferred from protocolStructured statistical methods
Our previous results raise the possibility that stimulating neurogenesis by fluoxetine may facilitate the regulation of stress systems. We therefore assessed whether UCMS and fl...; To examine more deeply the link between hippocampal neurogenesis and stress response, we investigated whether ablation of neurogenesis and UCMS induce abnormalities in the HPA a...; Considering that relatively small sample sizes were used and that assumptions for parametric statistics could not be ensured (normality and homoscedasticity), data were analyzed...; We first assessed the effects on the whole granule cell population (, P =0.0209). Although UCMS-vehicle mice exhibited an apparent lower NeuN+/Fos+ cell density than the other...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
The stress regimen used was previously described and is a variant of the chronic mild stress procedures described by Willner in rats. UCMS mice were repeatedly subjected to various socio-environmental stressors according to an unpredictable schedule for a total period of 6-8 weeks. From the third week of UCMS onward, mice were administered intraperitoneal (ip), once a day, with either vehicle, fluoxetine (20 mg kg -1 per day) or SSR125543 (20 mg kg -1 per day). Treatment was always maintained until the end of the experiments. Body weight and coat state were assessed weekly, as markers of the progression of the UCMS-evoked syndrome.,
This test required a device containing three aligned compartments with the same dimension (20 × 20 × 20 cm). Only the colors of the walls and the floor were different between the compartments (Figure 2a). Mice were first familiarized with a chocolate cookie (Pepito, Lu, France) 4.5 weeks before the first testing. One hour before testing, the regular food was removed from the cage lid. At the time of testing, a small amount (2±1 g) of chocolate cookie (or regular food in a control experiment) was placed at the center of the black compartment. The mouse was initially placed in the white compartment of the apparatus. Each session of the test lasted five minutes. The cookie consumption (# bites) was recorded within the 5 min test period. Four sessions of testing were performed within 10 days (inter-test interval: 2 days).
DEX suppression test: mice were i.p. injected with either DEX-P (0.1 mg kg -1, five mice per group) or saline (0.9% NaCl, five mice per group). All fecalthe boli that were voided between 8 and 10 h after the injection were collected to measure the level of fecal corticosterone metabolites.
Intrahippocampal DEX infusion: the infusion procedure was derived from the method previously described. Mice were stereotaxically and bilaterally implanted with guide cannulas (6 mm long, 0.6 mm outer diameter, 0.36 mm inner diameter). The following coordinates were used for the guide cannula implantation: bregma=-3.08, lateral=±2.3, vertical=-1.4. After 1-week recovery period from the surgery, infusion cannulas (7 mm long, 0.3 mm outer diameter, 0.17 mm inner diameter) were placed and extended 1 mm below the guide cannulas. The animals received bilateral infusions during 1.5 min of either vehicle (0.9% NaCl/0.2% ethanol) or DEX (50 ng in 0.6 µl). In one experiment, the brain was collected 2 h following the infusion to assess DEX-induced Fos changes in downstream brain structures and the PVN (Figure 3). In another experiment, the animals were killed by CO 2 asphyxiation 4 h following infusion and trunk blood was collected to measure plasma corticosterone levels (Figure 6).
We then directly tested the ability of the DG granule neurons to react to glucocorticoids. These cells can be activated and express immediate early genes when individuals are exposed to a new environment.,, We took advantage of this property to examine the sensitivity of new and older granule cells to a new environment exposure following a single injection of DEX-P or vehicle ( ). For this purpose, we concurrently labeled cells for the neuronal marker NeuN, the immediate early gene product Fos ('activated' granule cells) and the proliferation marker BrdU (4 week-old newborn cells) ( ).
Machine-readable layer
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