Spatial Relational Memory Requires Hippocampal Adult Neurogenesis methods
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Fluorescence Resonance Energy Transfer
FRET analysis of Bax multimers was used to track the intermolecular interaction between ECFPBax and EYFPBax fusion proteins. For cultured CHO cells, FRET was quantified using "Microfret". In brief, this method consists in the acquisition of three images through i) a CFP filter set (excitation 436/10 nm, emission 470/30 nm); ii) a YFP filter set (excitation 500/20 nm, emission 535/30 nm); iii) a FRET filter set (excitation 436/10 nm, emission 535/30 nm), and the determination of the "corrected FRET" or FRETc value which takes into consideration the non-FRET signal caused by the overlapping of the CFP and YFP spectra. Thus, the calculation of FRETc depends on the following relationship: FRETc = IFRET-CCFP-CYFP, where IFRET corresponds to the intensity of FRET with the FRET filter set of CFP/YFP-coexpressing cells; CCFP and CYFP correspond...
Behavioral analysis
At 14 weeks of age, 6 weeks after the beginning of the Dox treatment, independent groups of mice were tested in two separate experiments (experiments 2 and 3) for three behavioral tasks, each task being separated by a one week interval. The order of the tasks in the two experiments was changed in order to control for the possible bias due to interactions between tasks or treatment duration ( ). In experiment 2, novel environment exploration was tested twice, the first time at the beginning and the second time at the end of the behavioral sequence. Animals were then tested in a water maze followed by a fear conditioning test. In experiment 3 the opposite sequence was used. During water maze testing, in experiment 2 animals were first trained with a variable starting-point procedure, followed by a probe test, a visible platform test, and then by a constant starting point procedure befor...
Training with constant start positions
During this test mice were also required to locate the hidden platform using distal extra-maze cues. Procedures were similar to the ones used for training with variable start positions (3 daily trials with a 60 seconds cut-off and 5 min inter-trial intervals). However this time the start position was maintained constant between trials and days of training. Also the hidden platform was localized in a different quadrant than the one used for the variable-start paradigm. When performances reached a criterion of less than 10 sec of escape latency over two successive trials (2 nd day of training in experiment 2; 8 th day of training in experiment 3), animals were tested to locate the hidden platform from a novel start position that had never been used before (1 trial with a 60 seconds cut-off).
Hippocampal inactivation
In order to verify the hippocampal dependency of our fear conditioning procedure, supplementary groups of C57BL/6J male animals (n = 13) were implanted with bilateral hippocampal cannulae, and were injected with either lidocaine or vehicle solutions just before the conditioning session. Stainless-steel guided cannulae (26 gauge, 8 mm in length) were implanted bilaterally 1 mm above the dorsal hippocampus (A/P, -2000 µm; M/L, ±1300 µm; D/V, 1000 µm). Guides were permanently fixed to the skull with dental cement and two jewel screws and were obturated by appropriate stylets until behavioral manipulations. After surgery, mice were allowed to recover for at least 2 weeks. For infusions, the stylets obtruding the guide cannulae were removed. Stainless-steel cannulae (32 gauge, 9 mm) attached to 1 µl Hamilton syringes (PolyLabo, Strasbourg, France)...
Immunohistochemistry
At the end of experiments, mice were perfused transcardially with a phosphate-buffered solution of 4% paraformaldehyde and brains were cut on a vibratome (40 µm thick sections). For each staining, one in ten free-floating sections was processed according to a standard immunohistochemical procedure. HH3 immunoreactivity (IR), nestin-IR, activated caspase 3-IR, Dcx-IR, CD11b-IR or PECAM-1-IR were revealed using respectively a rabbit anti-HH3 polyclonal antibody (1∶2000, Upstate), a mouse anti-nestin monoclonal antibody (1∶1000; Chemicon), a rabbit anti-activated caspase 3 polyclonal antibody (1:500, Cell signaling technology), a sheep anti-Dcx polyclonal antibody (1∶2000; SantaCruz), a rat anti-mouse CD11b monoclonal antibody (1∶1000; Serotec) and a rat anti-mouse PECAM-1 monoclonal antibody (1:500; Pharmingen). For BrdU labeling, sections were treated with...
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Evidence quotes (5)
FRET analysis of Bax multimers was used to track the intermolecular interaction between ECFPBax and EYFPBax fusion proteins. For cultured CHO cells, FRET was quantified using "Microfret". In brief, this method consists in the acquisition of three images through i) a CFP filter set (excitation 436/10 nm, emission 470/30 nm); ii) a YFP filter set (excitation 500/20 nm, emission 535/30 nm); iii) a FRET filter set (excitation 436/10 nm, emission 535/30 nm), and the determination of the "corrected FRET" or FRETc value which takes into consideration the non-FRET signal caused by the overlapping of the CFP and YFP spectra. Thus, the calculation of FRETc depends on the following relationship: FRETc = IFRET-CCFP-CYFP, where IFRET corresponds to the intensity of FRET with the FRET filter set of CFP/YFP-coexpressing cells; CCFP and CYFP correspond respectively to the crosstalk between CFP and YFP due to overlapping spectra. This phenomenon was evaluated in cells expressing only one of the two constructs, because the contribution of CFP and YFP to FRET in coexpressed cells is proportional to that in singly expressed cells. The right side of t...
At 14 weeks of age, 6 weeks after the beginning of the Dox treatment, independent groups of mice were tested in two separate experiments (experiments 2 and 3) for three behavioral tasks, each task being separated by a one week interval. The order of the tasks in the two experiments was changed in order to control for the possible bias due to interactions between tasks or treatment duration ( ). In experiment 2, novel environment exploration was tested twice, the first time at the beginning and the second time at the end of the behavioral sequence. Animals were then tested in a water maze followed by a fear conditioning test. In experiment 3 the opposite sequence was used. During water maze testing, in experiment 2 animals were first trained with a variable starting-point procedure, followed by a probe test, a visible platform test, and then by a constant starting point procedure before finally testing them with a novel start point. In experiment 3 the inverse order of water maze paradigms was used: animals were first trained with a constant starting-point procedure followed by testing with a novel starting point, and then a variable starting point procedure followed by a probe t...
During this test mice were also required to locate the hidden platform using distal extra-maze cues. Procedures were similar to the ones used for training with variable start positions (3 daily trials with a 60 seconds cut-off and 5 min inter-trial intervals). However this time the start position was maintained constant between trials and days of training. Also the hidden platform was localized in a different quadrant than the one used for the variable-start paradigm. When performances reached a criterion of less than 10 sec of escape latency over two successive trials (2 nd day of training in experiment 2; 8 th day of training in experiment 3), animals were tested to locate the hidden platform from a novel start position that had never been used before (1 trial with a 60 seconds cut-off).
In order to verify the hippocampal dependency of our fear conditioning procedure, supplementary groups of C57BL/6J male animals (n = 13) were implanted with bilateral hippocampal cannulae, and were injected with either lidocaine or vehicle solutions just before the conditioning session. Stainless-steel guided cannulae (26 gauge, 8 mm in length) were implanted bilaterally 1 mm above the dorsal hippocampus (A/P, -2000 µm; M/L, ±1300 µm; D/V, 1000 µm). Guides were permanently fixed to the skull with dental cement and two jewel screws and were obturated by appropriate stylets until behavioral manipulations. After surgery, mice were allowed to recover for at least 2 weeks. For infusions, the stylets obtruding the guide cannulae were removed. Stainless-steel cannulae (32 gauge, 9 mm) attached to 1 µl Hamilton syringes (PolyLabo, Strasbourg, France) with polyethylene catheter tubing were inserted through the guide cannulae. The syringes were fixed in a constant rate infusion pump. Each mouse was given bilateral infusions of 0.9% saline (Vehicle, Veh, 0.3µl/side) or 4% Lidocaine (Lido, Sigma, 0.3µl/side) into the dorsal hippocampus. I...
At the end of experiments, mice were perfused transcardially with a phosphate-buffered solution of 4% paraformaldehyde and brains were cut on a vibratome (40 µm thick sections). For each staining, one in ten free-floating sections was processed according to a standard immunohistochemical procedure. HH3 immunoreactivity (IR), nestin-IR, activated caspase 3-IR, Dcx-IR, CD11b-IR or PECAM-1-IR were revealed using respectively a rabbit anti-HH3 polyclonal antibody (1∶2000, Upstate), a mouse anti-nestin monoclonal antibody (1∶1000; Chemicon), a rabbit anti-activated caspase 3 polyclonal antibody (1:500, Cell signaling technology), a sheep anti-Dcx polyclonal antibody (1∶2000; SantaCruz), a rat anti-mouse CD11b monoclonal antibody (1∶1000; Serotec) and a rat anti-mouse PECAM-1 monoclonal antibody (1:500; Pharmingen). For BrdU labeling, sections were treated with 2N HCl (30 min at 37°C) and then incubated with a rat monoclonal anti-BrdU antibody (1∶2000, Accurate, New York, USA). For each staining, sections were processed in parallel and immunoreactivities were visualized by the biotin-streptavidin technique (ABC kit, Dako) using 3,3′-diami...
Machine-readable layer
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