Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis methods
Aim. Evidence-backed execution summary for Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis methods from Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
BDNF cloning
reagent used in the protocol.
- Use
- Rat total DNA was extracted from a C6 rat glioma cell line using the GenElute mammalian genomic DNA purification kit (Sigma-Aldrich, St Louis, MO, USA), and the coding sequence of the BDNF gene was subsequently amplified by PCR using an Expand High Fidelity DNA polymerase (Roche Diagnostics, Penzberg, Germany). The...
Effectiveness of the various shRNA sequences in vitro
reagent used in the protocol.
- Use
- 293T cells were cultured with Dulbecco's modified eagle's medium (Gibco-BRL, Invitrogen, Belgium) containing 10% fetal calf serum (GIBCO-BRL). Twenty-four hours before transfection, 293T cells were split 1:5 to reach 60% confluency. A quantity of 8.4 µg of either one or a mix of the shRNA vectors (sh1-sh4...
Cloning lentiviral silencing vectors
reagent used in the protocol.
- Use
- Complete H1 promoter and shBDNF, or shSCR cassettes were amplified from pSilencer plasmid by PCR using Expand High Fidelity DNA polymerase and the following primers: 5′-GCGCTCGAGGTTTTCCCAGTCACGAC-3′ (forward) and 5′-ATCGAGTTAGCTCACTCATTAGGC-3′ (reverse). The forward primer was designed to inc...
High titer lentiviral preparation
reagent used in the protocol.
- Use
- 293T cells were split into 15 cm plates, precoated with 20 µg ml -1 of Poly-L-lysin (Sigma-Aldrich, USA), to reach 60% confluency. Cells were transfected using polyethyleneimine as described previously. In brief, on the transfection day, medium was replaced with Dulbecco's modified eagle...
Validation of LV-shBDNF activity in vitro
reagent used in the protocol.
- Use
- C6 rat glioma cells were grown to 60% confluency and infected with LV-shBDNF, LV-shSCR or LV-GFP supplemented with polybrene (8 µg ml -1 ). Cells were allowed to grow to 100% confluency and medium was replaced. Twenty-four hours later, medium was collected and stored at -20 °C...
Behavioral effects of BDNF knockdown in the vSUB
reagent used in the protocol.
- Use
- Brain-derived neurotrophic factor knockdown in the vSUB reduced sucrose preference significantly as compared with control animals when sucrose concentration was 0.2% ( ). However, this reduction was not significant when rats were tested using a 2% sucrose solution ( ). Activity in FST or home-cage locomotion was not...
Sucrose preference test
reagent used in the protocol.
- Use
- Sucrose preference was performed as we have previously described., Rats had access to two drinking spouts positioned side-by-side at the rear of the cage. Fluid consumption was recorded by weighing the bottles every morning between 0930 and 1000 h. Sucrose solution (prepared in tap water) was placed in one bo...
Sucrose preference test
reagent used in the protocol.
- Use
- To increase the sensitivity of this method, different concentrations of sucrose solution were measured in order to examine which concentration of sucrose could reliably be distinguished ( ). On the basis of dose-response curve, we chose a high concentration (2%) at which rats strongly preferred the sucrose sol...
Forced swim test
A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, The water temperature was kept at 24 °C (2 °C above room temperature) and the water level was such that the rat could no...
- Use
- A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, The water temperature was kept at 24 °C (2 °C above room temperature) and the water level was such that the rat could no...
Effectiveness of the various shRNA sequences in vitro
293T cells were cultured with Dulbecco's modified eagle's medium (Gibco-BRL, Invitrogen, Belgium) containing 10% fetal calf serum (GIBCO-BRL). Twenty-four hours before transfection, 293T cells were split 1:5 to reach 60% confluency. A quantity of 8.4 µg of either one or a mix of the shRNA vectors (sh1-sh4...
- Use
- 293T cells were cultured with Dulbecco's modified eagle's medium (Gibco-BRL, Invitrogen, Belgium) containing 10% fetal calf serum (GIBCO-BRL). Twenty-four hours before transfection, 293T cells were split 1:5 to reach 60% confluency. A quantity of 8.4 µg of either one or a mix of the shRNA vectors (sh1-sh4...
Validation of LV-shBDNF activity in vitro
C6 rat glioma cells were grown to 60% confluency and infected with LV-shBDNF, LV-shSCR or LV-GFP supplemented with polybrene (8 µg ml -1 ). Cells were allowed to grow to 100% confluency and medium was replaced. Twenty-four hours later, medium was collected and stored at -20 °C...
- Use
- C6 rat glioma cells were grown to 60% confluency and infected with LV-shBDNF, LV-shSCR or LV-GFP supplemented with polybrene (8 µg ml -1 ). Cells were allowed to grow to 100% confluency and medium was replaced. Twenty-four hours later, medium was collected and stored at -20 °C...
Behavioral effects of BDNF knockdown in the vSUB
Brain-derived neurotrophic factor knockdown in the vSUB reduced sucrose preference significantly as compared with control animals when sucrose concentration was 0.2% ( ). However, this reduction was not significant when rats were tested using a 2% sucrose solution ( ). Activity in FST or home-cage locomotion was not...
- Use
- Brain-derived neurotrophic factor knockdown in the vSUB reduced sucrose preference significantly as compared with control animals when sucrose concentration was 0.2% ( ). However, this reduction was not significant when rats were tested using a 2% sucrose solution ( ). Activity in FST or home-cage locomotion was not...
Microinjection of LV-shRNA
A week after surgery, rats were evaluated for their sucrose preference levels (see section 'Sucrose preference test' for description) and divided into two groups with similar averaged preference. Two microliters of either LV-shBDNF (treatment) or LV-shSCR (control) were microinjected bilaterally into the speci...
- Use
- A week after surgery, rats were evaluated for their sucrose preference levels (see section 'Sucrose preference test' for description) and divided into two groups with similar averaged preference. Two microliters of either LV-shBDNF (treatment) or LV-shSCR (control) were microinjected bilaterally into the speci...
Home-cage locomotion
Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on infrared sensors located above the home cage of each rat. Mobility during the dark period (over 12 h per night) was measured for 5 days....
- Use
- Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on infrared sensors located above the home cage of each rat. Mobility during the dark period (over 12 h per night) was measured for 5 days....
Exploration and novelty-induced behavior
Locomotion was performed as we have previously described., Rats were placed in a 40 × 40 cm exploration box (ActiMot System Activity Chamber, TSE). Then, distance traveled and number of rearings were recorded automatically over 10 min. The distance traveled was estimated by beam breaks of 32 photo...
- Use
- Locomotion was performed as we have previously described., Rats were placed in a 40 × 40 cm exploration box (ActiMot System Activity Chamber, TSE). Then, distance traveled and number of rearings were recorded automatically over 10 min. The distance traveled was estimated by beam breaks of 32 photo...
Morris water maze
The Morris water maze was performed as we described previously. Briefly, rats were trained and tested for 4 consecutive days and tested again on the fifth day. In the training sessions, rats were placed in a water tank (diameter of 1.4 m) with a hidden platform placed 1.5 cm below the water surface. Visu...
- Use
- The Morris water maze was performed as we described previously. Briefly, rats were trained and tested for 4 consecutive days and tested again on the fifth day. In the training sessions, rats were placed in a water tank (diameter of 1.4 m) with a hidden platform placed 1.5 cm below the water surface. Visu...
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Forced swim test
A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, The water temperature was kept at 24 °C (2 °C above room temperature) and the water level was such that the rat could not touch the bottom with its hind paws. Rats were given a single 10 min exposure to the swim tank and were videotaped. Video films of each FST session were carefully analyzed by an observer blind to the treatment groups, using our locally developed software for a computer-attached joystick. The computerized analysis for FST was developed in our laboratory to continuously follow the limbs of the rats and detect fine alterations in mobility throughout the test. This allowed for a sensitive analysis of more information than the standard scoring protocols. In addition, imm...
High titer lentiviral preparation
293T cells were split into 15 cm plates, precoated with 20 µg ml -1 of Poly-L-lysin (Sigma-Aldrich, USA), to reach 60% confluency. Cells were transfected using polyethyleneimine as described previously. In brief, on the transfection day, medium was replaced with Dulbecco's modified eagle's medium (which did not include fetal calf serum). DNA mixture containing 26 µg of transfer plasmid, 16.9 µg of packaging plasmid (encoding viral Gag and Pol proteins (pCMVΔR8.91)), and 9.1 µg of envelope plasmid (encoding the envelope of vesicular stomatitis virus (pCI)) and 78 µl of 1 mg ml -1 polyethyleneimine in a total volume of 1.5 ml Dulbecco's modified eagle's medium was prepared. The mixture was incubated for 10 min at room temperature and then added drop wise to plates containing 293T...
Intracranial surgery
Rats were anesthetized with ketamine (170 mg kg -1 ) and acepromazine (1.7 mg kg -1 ) and placed in a stereotaxic frame. Stainless steel guide cannulae (Plastics One, Roanoke, VA, USA) were implanted bilaterally into either the dorsal dentate gyrus (dDG) ( n =61), CA3 ( n =18) or ventral subiculum (vSUB) ( n =22) subregions of the hippocampus using the coordinates described below (relative to bregma and according to the atlas of Paxinos and Watson, 1998). dDG: -3.8 anteroposterior, +2.4 mediolateral and -1.95 dorsoventral (from dura) at a lateral angle of 10° CA3: -3.8 anteroposterior, 4.47 mediolateral and -1.36 dorsoventral (from dura) at a lateral angle of 10° vSUB: -6.5 anteroposterior, +7.05 mediolateral and -3.7 dorsoventral (from dura) at a lateral angle of 16°.
Microinjection of LV-shRNA
A week after surgery, rats were evaluated for their sucrose preference levels (see section 'Sucrose preference test' for description) and divided into two groups with similar averaged preference. Two microliters of either LV-shBDNF (treatment) or LV-shSCR (control) were microinjected bilaterally into the specific hippocampal subregions over 4 min using a dual channel MAB 40 microdialysis pump (Microbiotech/SE AB, Stockholm, Sweden). Injectors were left in place for 2 min after completing the injection to ensure adequate diffusion from the injector tip. Microinjections were repeated three times every other day. One week after the last injection, the behavioral testing was initiated. All the behavioral assessments described below were performed for each rat, in the same order as written below.
Sucrose preference test
Sucrose preference was performed as we have previously described., Rats had access to two drinking spouts positioned side-by-side at the rear of the cage. Fluid consumption was recorded by weighing the bottles every morning between 0930 and 1000 h. Sucrose solution (prepared in tap water) was placed in one bottle and tap water in the other. Rats were tested for 4 days, followed by 1 day of only tap water and then bottle positions were switched and rats were tested for an additional 4 days to control for side preference. Sucrose preference for each rat was defined as the average percentage of sucrose consumption of the total liquid consumption.
Sucrose preference test
To increase the sensitivity of this method, different concentrations of sucrose solution were measured in order to examine which concentration of sucrose could reliably be distinguished ( ). On the basis of dose-response curve, we chose a high concentration (2%) at which rats strongly preferred the sucrose solution, and a low concentration (0.2%) at which rats show a significant, but moderate sucrose preference over plain water. We have previously shown that this method has increased sensitivity and allows for measurement of more subtle, but significant reductions of sucrose preference when applied to the chronic mild stress model for depression.,
Home-cage locomotion
Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on infrared sensors located above the home cage of each rat. Mobility during the dark period (over 12 h per night) was measured for 5 days. Increasing the measurement time in untouched animals in their home cages increases the stability and reproducibility of locomotion data and therefore enhances the probability of observing significant and replicable behavioral changes.
Morris water maze
BrdU administration: Following the behavioral tests, rats were injected intraperitoneally with 50 mg kg -1 bromodeoxyuridine (BrdU; Sigma-Aldrich) at 12 h intervals for 2 days. Twenty four hours or a week after the last BrdU injection, rats were deeply anesthetized and transcardially perfused using PBS supplemented with 4 U ml -1 heparin (Merck Biosciences, Darmstadt, Germany), followed by fresh 2.5 % paraformaldehyde (J.T. Baker, Deventer, Holland) supplemented with 5% sucrose (J.T. Baker) in PBS. Brains were removed, postfixed overnight, and allowed to sink in 2.5% paraformaldehyde supplemented with 30% sucrose in PBS solution. The coronal sections (30 µm) were collected using a cryostat (Leica CM 3050 S, Nussloch, Germany) and placed into 24-well plates containing 0.01% azide (Sigma-Aldrich) in PBS and were stored at 4 ...
Measurement outputs
What raw and processed outputs should exist?
A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, Th...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Rat total DNA was extracted from a C6 rat glioma cell line using the GenElute mammalian genomic DNA purification kit (Sigma-Aldrich, St Louis, MO, USA), and the coding sequence...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
293T cells were cultured with Dulbecco's modified eagle's medium (Gibco-BRL, Invitrogen, Belgium) containing 10% fetal calf serum (GIBCO-BRL). Twenty-four hours before transfect...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Complete H1 promoter and shBDNF, or shSCR cassettes were amplified from pSilencer plasmid by PCR using Expand High Fidelity DNA polymerase and the following primers: 5′-GC...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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
Brain-derived neurotrophic factor knockdown in the vSUB reduced sucrose preference significantly as compared with control animals when sucrose concentration was 0.2% ( ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, Th...; Rat total DNA was extracted from a C6 rat glioma cell line using the GenElute mammalian genomic DNA purification kit (Sigma-Aldrich, St Louis, MO, USA), and the coding sequence...; 293T cells were cultured with Dulbecco's modified eagle's medium (Gibco-BRL, Invitrogen, Belgium) containing 10% fetal calf serum (GIBCO-BRL). Twenty-four hours before transfect...; Complete H1 promoter and shBDNF, or shSCR cassettes were amplified from pSilencer plasmid by PCR using Expand High Fidelity DNA polymerase and the following primers: 5′-GC....
from paperStatistical comparison
Brain-derived neurotrophic factor knockdown in the vSUB reduced sucrose preference significantly as compared with control animals when sucrose concentration was 0.2% ( ). Howeve...; To increase the sensitivity of this method, different concentrations of sucrose solution were measured in order to examine which concentration of sucrose could reliably be disti...; Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on inf...; Results are expressed as mean±s.e.m. A two-tailed unpaired Student's t -test was used for analyses of the behavioral experiments, except for the learning curve of the Morri...
from paperReporting output
Report representative outputs alongside summary comparisons for A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, Th..., Rat total DNA was extracted from a C6 rat glioma cell line using the GenElute mammalian genomic DNA purification kit (Sigma-Aldrich, St Louis, MO, USA), and the coding sequence..., 293T cells were cultured with Dulbecco's modified eagle's medium (Gibco-BRL, Invitrogen, Belgium) containing 10% fetal calf serum (GIBCO-BRL). Twenty-four hours before transfect..., Complete H1 promoter and shBDNF, or shSCR cassettes were amplified from pSilencer plasmid by PCR using Expand High Fidelity DNA polymerase and the following primers: 5′-GC....
inferred from protocolStructured statistical methods
Brain-derived neurotrophic factor knockdown in the vSUB reduced sucrose preference significantly as compared with control animals when sucrose concentration was 0.2% ( ). Howeve...; To increase the sensitivity of this method, different concentrations of sucrose solution were measured in order to examine which concentration of sucrose could reliably be disti...; Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on inf...; Results are expressed as mean±s.e.m. A two-tailed unpaired Student's t -test was used for analyses of the behavioral experiments, except for the learning curve of the Morri...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, The water temperature was kept at 24 °C (2 °C above room temperature) and the water level was such that the rat could not touch the bottom with its hind paws. Rats were given a single 10 min exposure to the swim tank and were videotaped. Video films of each FST session were carefully analyzed by an observer blind to the treatment groups, using our locally developed software for a computer-attached joystick. The computerized analysis for FST was developed in our laboratory to continuously follow the limbs of the rats and detect fine alterations in mobility throughout the test. This allowed for a sensitive analysis of more information than the standard scoring protocols. In addition, immobility time was measured manually.
293T cells were split into 15 cm plates, precoated with 20 µg ml -1 of Poly-L-lysin (Sigma-Aldrich, USA), to reach 60% confluency. Cells were transfected using polyethyleneimine as described previously. In brief, on the transfection day, medium was replaced with Dulbecco's modified eagle's medium (which did not include fetal calf serum). DNA mixture containing 26 µg of transfer plasmid, 16.9 µg of packaging plasmid (encoding viral Gag and Pol proteins (pCMVΔR8.91)), and 9.1 µg of envelope plasmid (encoding the envelope of vesicular stomatitis virus (pCI)) and 78 µl of 1 mg ml -1 polyethyleneimine in a total volume of 1.5 ml Dulbecco's modified eagle's medium was prepared. The mixture was incubated for 10 min at room temperature and then added drop wise to plates containing 293T cells. After 16 h transfection, the medium was replaced with Dulbecco's modified eagle's medium supplemented with 10% fetal calf serum. Forty eight and seventy two hours after transfection, medium was collected, cleared by low speed centrifugation, filtered through 0.45 µm-pore-size...
Rats were anesthetized with ketamine (170 mg kg -1 ) and acepromazine (1.7 mg kg -1 ) and placed in a stereotaxic frame. Stainless steel guide cannulae (Plastics One, Roanoke, VA, USA) were implanted bilaterally into either the dorsal dentate gyrus (dDG) ( n =61), CA3 ( n =18) or ventral subiculum (vSUB) ( n =22) subregions of the hippocampus using the coordinates described below (relative to bregma and according to the atlas of Paxinos and Watson, 1998). dDG: -3.8 anteroposterior, +2.4 mediolateral and -1.95 dorsoventral (from dura) at a lateral angle of 10° CA3: -3.8 anteroposterior, 4.47 mediolateral and -1.36 dorsoventral (from dura) at a lateral angle of 10° vSUB: -6.5 anteroposterior, +7.05 mediolateral and -3.7 dorsoventral (from dura) at a lateral angle of 16°.
A week after surgery, rats were evaluated for their sucrose preference levels (see section 'Sucrose preference test' for description) and divided into two groups with similar averaged preference. Two microliters of either LV-shBDNF (treatment) or LV-shSCR (control) were microinjected bilaterally into the specific hippocampal subregions over 4 min using a dual channel MAB 40 microdialysis pump (Microbiotech/SE AB, Stockholm, Sweden). Injectors were left in place for 2 min after completing the injection to ensure adequate diffusion from the injector tip. Microinjections were repeated three times every other day. One week after the last injection, the behavioral testing was initiated. All the behavioral assessments described below were performed for each rat, in the same order as written below.
Sucrose preference was performed as we have previously described., Rats had access to two drinking spouts positioned side-by-side at the rear of the cage. Fluid consumption was recorded by weighing the bottles every morning between 0930 and 1000 h. Sucrose solution (prepared in tap water) was placed in one bottle and tap water in the other. Rats were tested for 4 days, followed by 1 day of only tap water and then bottle positions were switched and rats were tested for an additional 4 days to control for side preference. Sucrose preference for each rat was defined as the average percentage of sucrose consumption of the total liquid consumption.
To increase the sensitivity of this method, different concentrations of sucrose solution were measured in order to examine which concentration of sucrose could reliably be distinguished ( ). On the basis of dose-response curve, we chose a high concentration (2%) at which rats strongly preferred the sucrose solution, and a low concentration (0.2%) at which rats show a significant, but moderate sucrose preference over plain water. We have previously shown that this method has increased sensitivity and allows for measurement of more subtle, but significant reductions of sucrose preference when applied to the chronic mild stress model for depression.,
Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on infrared sensors located above the home cage of each rat. Mobility during the dark period (over 12 h per night) was measured for 5 days. Increasing the measurement time in untouched animals in their home cages increases the stability and reproducibility of locomotion data and therefore enhances the probability of observing significant and replicable behavioral changes.
BrdU administration: Following the behavioral tests, rats were injected intraperitoneally with 50 mg kg -1 bromodeoxyuridine (BrdU; Sigma-Aldrich) at 12 h intervals for 2 days. Twenty four hours or a week after the last BrdU injection, rats were deeply anesthetized and transcardially perfused using PBS supplemented with 4 U ml -1 heparin (Merck Biosciences, Darmstadt, Germany), followed by fresh 2.5 % paraformaldehyde (J.T. Baker, Deventer, Holland) supplemented with 5% sucrose (J.T. Baker) in PBS. Brains were removed, postfixed overnight, and allowed to sink in 2.5% paraformaldehyde supplemented with 30% sucrose in PBS solution. The coronal sections (30 µm) were collected using a cryostat (Leica CM 3050 S, Nussloch, Germany) and placed into 24-well plates containing 0.01% azide (Sigma-Aldrich) in PBS and were stored at 4 °C until further use.
Machine-readable layer
[
{
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"@type": "HowTo",
"name": "Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis methods",
"description": "Evidence-backed execution summary for Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis methods from Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis.",
"totalTime": "PT5280M",
"step": [
{
"@type": "HowToStep",
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"name": "Forced swim test",
"text": "A modified FST was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at the Weizmann Institute), as we have previously described.,, The water temperature was kept at 24 °C (2 °C above room temperature) and the water level was such that the rat could not touch the bottom with its hind paws. Rats were given a single 10 min exposure to the swim tank and were videotaped. Video films of each FST session were carefully analyzed by an observer blind to the treatment groups, using our locally developed software for a computer-attached joystick. The computerized analysis for FST was developed in our laboratory to continuously follow the limbs of the rats and detect fine alterations in mobility throughout the test. This allowed for a sensitive analysis of more information than the standard scoring protocols. In addition, imm..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "High titer lentiviral preparation",
"text": "293T cells were split into 15 cm plates, precoated with 20 µg ml -1 of Poly-L-lysin (Sigma-Aldrich, USA), to reach 60% confluency. Cells were transfected using polyethyleneimine as described previously. In brief, on the transfection day, medium was replaced with Dulbecco's modified eagle's medium (which did not include fetal calf serum). DNA mixture containing 26 µg of transfer plasmid, 16.9 µg of packaging plasmid (encoding viral Gag and Pol proteins (pCMVΔR8.91)), and 9.1 µg of envelope plasmid (encoding the envelope of vesicular stomatitis virus (pCI)) and 78 µl of 1 mg ml -1 polyethyleneimine in a total volume of 1.5 ml Dulbecco's modified eagle's medium was prepared. The mixture was incubated for 10 min at room temperature and then added drop wise to plates containing 293T..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Intracranial surgery",
"text": "Rats were anesthetized with ketamine (170 mg kg -1 ) and acepromazine (1.7 mg kg -1 ) and placed in a stereotaxic frame. Stainless steel guide cannulae (Plastics One, Roanoke, VA, USA) were implanted bilaterally into either the dorsal dentate gyrus (dDG) ( n =61), CA3 ( n =18) or ventral subiculum (vSUB) ( n =22) subregions of the hippocampus using the coordinates described below (relative to bregma and according to the atlas of Paxinos and Watson, 1998). dDG: -3.8 anteroposterior, +2.4 mediolateral and -1.95 dorsoventral (from dura) at a lateral angle of 10° CA3: -3.8 anteroposterior, 4.47 mediolateral and -1.36 dorsoventral (from dura) at a lateral angle of 10° vSUB: -6.5 anteroposterior, +7.05 mediolateral and -3.7 dorsoventral (from dura) at a lateral angle of 16°."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Microinjection of LV-shRNA",
"text": "A week after surgery, rats were evaluated for their sucrose preference levels (see section 'Sucrose preference test' for description) and divided into two groups with similar averaged preference. Two microliters of either LV-shBDNF (treatment) or LV-shSCR (control) were microinjected bilaterally into the specific hippocampal subregions over 4 min using a dual channel MAB 40 microdialysis pump (Microbiotech/SE AB, Stockholm, Sweden). Injectors were left in place for 2 min after completing the injection to ensure adequate diffusion from the injector tip. Microinjections were repeated three times every other day. One week after the last injection, the behavioral testing was initiated. All the behavioral assessments described below were performed for each rat, in the same order as written below."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Sucrose preference test",
"text": "Sucrose preference was performed as we have previously described., Rats had access to two drinking spouts positioned side-by-side at the rear of the cage. Fluid consumption was recorded by weighing the bottles every morning between 0930 and 1000 h. Sucrose solution (prepared in tap water) was placed in one bottle and tap water in the other. Rats were tested for 4 days, followed by 1 day of only tap water and then bottle positions were switched and rats were tested for an additional 4 days to control for side preference. Sucrose preference for each rat was defined as the average percentage of sucrose consumption of the total liquid consumption."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Sucrose preference test",
"text": "To increase the sensitivity of this method, different concentrations of sucrose solution were measured in order to examine which concentration of sucrose could reliably be distinguished ( ). On the basis of dose-response curve, we chose a high concentration (2%) at which rats strongly preferred the sucrose solution, and a low concentration (0.2%) at which rats show a significant, but moderate sucrose preference over plain water. We have previously shown that this method has increased sensitivity and allows for measurement of more subtle, but significant reductions of sucrose preference when applied to the chronic mild stress model for depression.,"
},
{
"@type": "HowToStep",
"position": 7,
"name": "Home-cage locomotion",
"text": "Chronic monitoring of locomotion was performed in the home cage using a computerized Inframot system (TSE, Bad Homburg, Germany) as described previously,, which is based on infrared sensors located above the home cage of each rat. Mobility during the dark period (over 12 h per night) was measured for 5 days. Increasing the measurement time in untouched animals in their home cages increases the stability and reproducibility of locomotion data and therefore enhances the probability of observing significant and replicable behavioral changes."
},
{
"@type": "HowToStep",
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"name": "Morris water maze",
"text": "BrdU administration: Following the behavioral tests, rats were injected intraperitoneally with 50 mg kg -1 bromodeoxyuridine (BrdU; Sigma-Aldrich) at 12 h intervals for 2 days. Twenty four hours or a week after the last BrdU injection, rats were deeply anesthetized and transcardially perfused using PBS supplemented with 4 U ml -1 heparin (Merck Biosciences, Darmstadt, Germany), followed by fresh 2.5 % paraformaldehyde (J.T. Baker, Deventer, Holland) supplemented with 5% sucrose (J.T. Baker) in PBS. Brains were removed, postfixed overnight, and allowed to sink in 2.5% paraformaldehyde supplemented with 30% sucrose in PBS solution. The coronal sections (30 µm) were collected using a cryostat (Leica CM 3050 S, Nussloch, Germany) and placed into 24-well plates containing 0.01% azide (Sigma-Aldrich) in PBS and were stored at 4 \u0011..."
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