Adult-Generated Hippocampal Neurons Allow the Flexible Use of Spatially Precise Learning Strategies methods
Aim. Evidence-backed execution summary for Adult-Generated Hippocampal Neurons Allow the Flexible Use of Spatially Precise Learning Strategies methods from Adult-Generated Hippocampal Neurons Allow the Flexible Use of Spatially Precise Learning Strategies.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Drug treatment
reagent used in the protocol.
- Use
- Temozolomide (TMZ, Temodal®, SP Europe, Belgium) is an alkylating agent intended for the treatment of recurrent malignant glioma. In a dosage finding experiment we found a dose of 25 mg/kg body weight to be effective in suppressing adult neurogenesis by more than 80% after monocyclic (3 days) of treatment ( ).
Drug treatment
reagent used in the protocol.
- Use
- Consequently, to suppress adult neurogenesis, mice from the treatment group (TMZ) received injections of TMZ at 25 mg/kg (i.p., 2,5 mg/ml in 0.9% NaCl), whereas the control group (CTR) received sham injections of saline only. This regimen was given on the first three days of a week for 4 weeks to resemble paradigms...
Drug treatment
reagent used in the protocol.
- Use
- For adult-born granule cells to become recruitable the sequence of proliferation, differentiation and maturation requires approximately up to 28 days. Thus, suppressing adult neurogenesis for at least 4 weeks combined with a reconstitution period of 4 more weeks ensured that most of the cells borne immediately befor...
Drug treatment
reagent used in the protocol.
- Use
- It has recently been shown that 6-8 weeks old, adult generated granule cells become selectively recruited during acquisition of a spatial learning task. Therefore, our primary aim was to minimize the number of potentially recruitable adult-born neurons exactly at that time, when the new granule cells should b...
Immunohistochemistry
reagent used in the protocol.
- Use
- For visualization of BrdU incorporation, DNA was denatured in 2N HCL for 30 minutes at 37°C. Free floating sections were then rinsed in 0.1 M borate buffer, pH 8.5, and thoroughly washed in tris-buffered saline (TBS), pH 7.4. To block endogenous peroxidase reactions, sections were pretreated with 0.6% H 2 O 2....
Reversal learning in the Morris water maze
We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-treated mice performed significantly worse than controls for the entire experiment (latency, F(1,590) = 34.95, p<0.001, repeated m...
- Use
- We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-treated mice performed significantly worse than controls for the entire experiment (latency, F(1,590) = 34.95, p<0.001, repeated m...
Reversal learning in the Morris water maze
(A) Both groups successfully learned to navigate to the hidden goal, but TMZ treated mice needed longer to locate the hidden platform. While the TMZ group reached comparable latency times at the end of the first acquisition phase, mice with suppressed adult neurogenesis performed worse both transiently before and co...
- Use
- (A) Both groups successfully learned to navigate to the hidden goal, but TMZ treated mice needed longer to locate the hidden platform. While the TMZ group reached comparable latency times at the end of the first acquisition phase, mice with suppressed adult neurogenesis performed worse both transiently before and co...
Reversal learning in the Morris water maze
Swim speed of both groups did not differ throughout the experiment (data not shown). In the 1st trial of day 4 (1st probe trial), when the platform had been moved to the opposite quadrant, mice from both groups spent significantly more time in the previous goal quadrant than in the other quadrants, indicative of suc...
- Use
- Swim speed of both groups did not differ throughout the experiment (data not shown). In the 1st trial of day 4 (1st probe trial), when the platform had been moved to the opposite quadrant, mice from both groups spent significantly more time in the previous goal quadrant than in the other quadrants, indicative of suc...
Reversal learning in the Morris water maze
After reversal, further significant differences were found. As apparent in the heat maps visualizing the probability for the mice to be present at a given location, TMZ-treated mice took significantly longer to develop a clear preference for the first goal and persevered swimming to that platform position even after...
- Use
- After reversal, further significant differences were found. As apparent in the heat maps visualizing the probability for the mice to be present at a given location, TMZ-treated mice took significantly longer to develop a clear preference for the first goal and persevered swimming to that platform position even after...
Qualitative aspects of spatial learning
To assess the qualitative aspects of learning the water maze task we analyzed the respective search strategies displayed by the mice to locate the hidden platform ( ). Parameter-based classification of search patterns turned out to be reliable for both groups indicating that treated animals used identical basic prin...
- Use
- To assess the qualitative aspects of learning the water maze task we analyzed the respective search strategies displayed by the mice to locate the hidden platform ( ). Parameter-based classification of search patterns turned out to be reliable for both groups indicating that treated animals used identical basic prin...
Convolution analysis
Our finding that treated mice reached comparable performance levels at the end of the first acquisition phase raised the important question whether progression to the spatially most precise, allocentric strategies is mandatory for learning the reference memory version of the water maze. Alternatively, the task might...
- Use
- Our finding that treated mice reached comparable performance levels at the end of the first acquisition phase raised the important question whether progression to the spatially most precise, allocentric strategies is mandatory for learning the reference memory version of the water maze. Alternatively, the task might...
Drug treatment
It has recently been shown that 6-8 weeks old, adult generated granule cells become selectively recruited during acquisition of a spatial learning task. Therefore, our primary aim was to minimize the number of potentially recruitable adult-born neurons exactly at that time, when the new granule cells should b...
- Use
- It has recently been shown that 6-8 weeks old, adult generated granule cells become selectively recruited during acquisition of a spatial learning task. Therefore, our primary aim was to minimize the number of potentially recruitable adult-born neurons exactly at that time, when the new granule cells should b...
Hematology
Blood cell counts were obtained using a Beckman Coulter Ac-T Diff Hematology Analyzer with a software version for veterinary applications. After anesthetizing the animals with diethylether, blood samples were taken from the retro-orbital sinus. Blood samples were taken 1 day before, 1 day after and 4 weeks after tre...
- Use
- Blood cell counts were obtained using a Beckman Coulter Ac-T Diff Hematology Analyzer with a software version for veterinary applications. After anesthetizing the animals with diethylether, blood samples were taken from the retro-orbital sinus. Blood samples were taken 1 day before, 1 day after and 4 weeks after tre...
Electrophysiology
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All data were analyzed offline using TIDA software. Amplitudes of evoked field potentials were measured from an average of 4 peaks. Data were expressed as means±SEM and statistical comparison was done by repeated-measures ANOVA on the last 10 min (Aabel Software, Gigawiz). Significance level was set to p <0.05.
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Drug treatment
Temozolomide (TMZ, Temodal®, SP Europe, Belgium) is an alkylating agent intended for the treatment of recurrent malignant glioma. In a dosage finding experiment we found a dose of 25 mg/kg body weight to be effective in suppressing adult neurogenesis by more than 80% after monocyclic (3 days) of treatment ( ).
Drug treatment
Consequently, to suppress adult neurogenesis, mice from the treatment group (TMZ) received injections of TMZ at 25 mg/kg (i.p., 2,5 mg/ml in 0.9% NaCl), whereas the control group (CTR) received sham injections of saline only. This regimen was given on the first three days of a week for 4 weeks to resemble paradigms used for glioma treatment in humans ( ). Behavioral testing was performed 4 weeks after the final TMZ injection.
Reversal learning in the Morris water maze
We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-treated mice performed significantly worse than controls for the entire experiment (latency, F(1,590) = 34.95, p<0.001, repeated measures ANOVA), both TMZ-treated mice and controls (CTR) successfully learned to navigate to the hidden platform during the first three days ( ). At the end of this period there were no significant differences between the groups in path length and latency time to reach the hidden platform. Although TMZ-treated animals and controls generally succeeded in learning the MWM, significant differences appeared on day two of the acquisition phase regarding both latency and path length (, t(9) = 3.42 p<0.001 for latency and t(9) = 2.77, p<0.01 for path lengt...
Materials and Methods
For all experiments we used 6-8 weeks old female C57BL6 mice. To label adult generated cells animals received one single intraperitoneal injection of bromodeoxyuridine (BrdU, 50 µg/kg body weight, Sigma). Tissue preparation was done as described. The mice were kept at the animal facility of the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch, Germany. All local and federal regulations of animal welfare in research were followed. The experiment was approved by the responsible authority, Landesamt für Technische Sicherheit und Gesundheit (LaGetSi) Berlin.
Drug treatment
For adult-born granule cells to become recruitable the sequence of proliferation, differentiation and maturation requires approximately up to 28 days. Thus, suppressing adult neurogenesis for at least 4 weeks combined with a reconstitution period of 4 more weeks ensured that most of the cells borne immediately before onset of TMZ treatment would have been already used or eliminated by apoptosis. Using intercalating convalescence times, we minimized the risk of confounding side effects during behavioral testing.
Drug treatment
It has recently been shown that 6-8 weeks old, adult generated granule cells become selectively recruited during acquisition of a spatial learning task. Therefore, our primary aim was to minimize the number of potentially recruitable adult-born neurons exactly at that time, when the new granule cells should be of particularly high relevance for task acquisition. Because we suppressed proliferation for at least 4 weeks and behavioral testing began an additional 4 weeks later, at the time-point of the behavioral analysis the subpopulation of 5-7 week old adult generated neurons is primarily affected by TMZ. An immediate recovery of the stem cell niche seemed unlikely as it was found that after wiping out proliferation in the SGZ by irradiation reconstitution occurs only slow and on a prolonged timescale. Consequently, at the time, when the mice were learning the water maze...
Immunohistochemistry
For visualization of BrdU incorporation, DNA was denatured in 2N HCL for 30 minutes at 37°C. Free floating sections were then rinsed in 0.1 M borate buffer, pH 8.5, and thoroughly washed in tris-buffered saline (TBS), pH 7.4. To block endogenous peroxidase reactions, sections were pretreated with 0.6% H 2 O 2. Sections were incubated with primary antibody against BrdU in TBS supplemented with 0.1% TritonX-100 and 3% donkey serum (TBS-plus) at 4°C overnight. After rinsing the sections with TBS and a blocking step with TBS-plus, incubation with the biotinylated secondary antibody (1∶500, Dianova) in TBS-plus followed. ABC reagent (Vectastain Elite, Vector Laboratories) was applied for 1 hour at a concentration of 9 µl/ml per reagent. Diaminobenzidine (DAB, Sigma) was used as a chromogen at 0.25 mg/ml in TBS with 0.01% H 2 O 2 and 0.04% nickelchloride followed by ri...
Hematology
Blood cell counts were obtained using a Beckman Coulter Ac-T Diff Hematology Analyzer with a software version for veterinary applications. After anesthetizing the animals with diethylether, blood samples were taken from the retro-orbital sinus. Blood samples were taken 1 day before, 1 day after and 4 weeks after treatment and collected using EDTA coated MiniCollect® tubes (Greiner bio-one, Kremsmünster, Austria). A total of five animals were used.
Measurement outputs
What raw and processed outputs should exist?
(A) Examples of search strategies recognized by the classification algorithm used. (B) Both groups showed a clear progression towards increasingly hippocampus-dependent strategi...
- 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
We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-tr...
- 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
(A) Both groups successfully learned to navigate to the hidden goal, but TMZ treated mice needed longer to locate the hidden platform. While the TMZ group reached comparable lat...
- 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
Because the distribution of differences in strategy use appeared to reflect an underlying temporal pattern we looked for systematical changes in the respective contributions to...
- 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
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
(A) Examples of search strategies recognized by the classification algorithm used.
from paperScoring or quantification
Quantify the primary readouts for this experiment: (A) Examples of search strategies recognized by the classification algorithm used. (B) Both groups showed a clear progression towards increasingly hippocampus-dependent strategi...; We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-tr...; (A) Both groups successfully learned to navigate to the hidden goal, but TMZ treated mice needed longer to locate the hidden platform. While the TMZ group reached comparable lat...; Because the distribution of differences in strategy use appeared to reflect an underlying temporal pattern we looked for systematical changes in the respective contributions to....
from paperStatistical comparison
(A) Examples of search strategies recognized by the classification algorithm used. (B) Both groups showed a clear progression towards increasingly hippocampus-dependent strategi...; To assess hippocampal function at the systems level we investigated LTP in the DG and CA1. In the DG, LTP is physiologically inhibited by GABAergic interneurons. Still lacking t...; (A) In TMZ mice treatment prevented LTP in the dentate gyrus of hippocampal slices perfused with ACSF, but LTP was induced in control mice (ANOVA F(1,198) = 252.8, p...; We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-tr...
from paperReporting output
Report representative outputs alongside summary comparisons for (A) Examples of search strategies recognized by the classification algorithm used. (B) Both groups showed a clear progression towards increasingly hippocampus-dependent strategi..., We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-tr..., (A) Both groups successfully learned to navigate to the hidden goal, but TMZ treated mice needed longer to locate the hidden platform. While the TMZ group reached comparable lat..., Because the distribution of differences in strategy use appeared to reflect an underlying temporal pattern we looked for systematical changes in the respective contributions to....
inferred from protocolStructured statistical methods
(A) Examples of search strategies recognized by the classification algorithm used. (B) Both groups showed a clear progression towards increasingly hippocampus-dependent strategi...; To assess hippocampal function at the systems level we investigated LTP in the DG and CA1. In the DG, LTP is physiologically inhibited by GABAergic interneurons. Still lacking t...; (A) In TMZ mice treatment prevented LTP in the dentate gyrus of hippocampal slices perfused with ACSF, but LTP was induced in control mice (ANOVA F(1,198) = 252.8, p...; We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-tr...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Temozolomide (TMZ, Temodal®, SP Europe, Belgium) is an alkylating agent intended for the treatment of recurrent malignant glioma. In a dosage finding experiment we found a dose of 25 mg/kg body weight to be effective in suppressing adult neurogenesis by more than 80% after monocyclic (3 days) of treatment ( ).
Consequently, to suppress adult neurogenesis, mice from the treatment group (TMZ) received injections of TMZ at 25 mg/kg (i.p., 2,5 mg/ml in 0.9% NaCl), whereas the control group (CTR) received sham injections of saline only. This regimen was given on the first three days of a week for 4 weeks to resemble paradigms used for glioma treatment in humans ( ). Behavioral testing was performed 4 weeks after the final TMZ injection.
We trained mice on a reference memory version of the MWM task with 6 trials per day for 5 days and a new platform position (reversal) on the beginning of day 4. Although TMZ-treated mice performed significantly worse than controls for the entire experiment (latency, F(1,590) = 34.95, p<0.001, repeated measures ANOVA), both TMZ-treated mice and controls (CTR) successfully learned to navigate to the hidden platform during the first three days ( ). At the end of this period there were no significant differences between the groups in path length and latency time to reach the hidden platform. Although TMZ-treated animals and controls generally succeeded in learning the MWM, significant differences appeared on day two of the acquisition phase regarding both latency and path length (, t(9) = 3.42 p<0.001 for latency and t(9) = 2.77, p<0.01 for path length, Student's t -test).
For all experiments we used 6-8 weeks old female C57BL6 mice. To label adult generated cells animals received one single intraperitoneal injection of bromodeoxyuridine (BrdU, 50 µg/kg body weight, Sigma). Tissue preparation was done as described. The mice were kept at the animal facility of the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch, Germany. All local and federal regulations of animal welfare in research were followed. The experiment was approved by the responsible authority, Landesamt für Technische Sicherheit und Gesundheit (LaGetSi) Berlin.
For adult-born granule cells to become recruitable the sequence of proliferation, differentiation and maturation requires approximately up to 28 days. Thus, suppressing adult neurogenesis for at least 4 weeks combined with a reconstitution period of 4 more weeks ensured that most of the cells borne immediately before onset of TMZ treatment would have been already used or eliminated by apoptosis. Using intercalating convalescence times, we minimized the risk of confounding side effects during behavioral testing.
It has recently been shown that 6-8 weeks old, adult generated granule cells become selectively recruited during acquisition of a spatial learning task. Therefore, our primary aim was to minimize the number of potentially recruitable adult-born neurons exactly at that time, when the new granule cells should be of particularly high relevance for task acquisition. Because we suppressed proliferation for at least 4 weeks and behavioral testing began an additional 4 weeks later, at the time-point of the behavioral analysis the subpopulation of 5-7 week old adult generated neurons is primarily affected by TMZ. An immediate recovery of the stem cell niche seemed unlikely as it was found that after wiping out proliferation in the SGZ by irradiation reconstitution occurs only slow and on a prolonged timescale. Consequently, at the time, when the mice were learning the water maze task only very few (or no) new granule cells were available to be functionally integrated into existing circuits.
For visualization of BrdU incorporation, DNA was denatured in 2N HCL for 30 minutes at 37°C. Free floating sections were then rinsed in 0.1 M borate buffer, pH 8.5, and thoroughly washed in tris-buffered saline (TBS), pH 7.4. To block endogenous peroxidase reactions, sections were pretreated with 0.6% H 2 O 2. Sections were incubated with primary antibody against BrdU in TBS supplemented with 0.1% TritonX-100 and 3% donkey serum (TBS-plus) at 4°C overnight. After rinsing the sections with TBS and a blocking step with TBS-plus, incubation with the biotinylated secondary antibody (1∶500, Dianova) in TBS-plus followed. ABC reagent (Vectastain Elite, Vector Laboratories) was applied for 1 hour at a concentration of 9 µl/ml per reagent. Diaminobenzidine (DAB, Sigma) was used as a chromogen at 0.25 mg/ml in TBS with 0.01% H 2 O 2 and 0.04% nickelchloride followed by rinsing with tap water and TBS. The sections were mounted on gelatine-coated glass slides and coverslipped with Neomount.
Blood cell counts were obtained using a Beckman Coulter Ac-T Diff Hematology Analyzer with a software version for veterinary applications. After anesthetizing the animals with diethylether, blood samples were taken from the retro-orbital sinus. Blood samples were taken 1 day before, 1 day after and 4 weeks after treatment and collected using EDTA coated MiniCollect® tubes (Greiner bio-one, Kremsmünster, Austria). A total of five animals were used.
Machine-readable layer
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