Adult-Generated Hippocampal Neurons Allow the Flexible Use of Spatially Precise Learning Strategies methods
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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.
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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.
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