Spatial Learning Depends on Both the Addition and Removal of New Hippocampal Neurons methods
Aim. Evidence-backed execution summary for Spatial Learning Depends on Both the Addition and Removal of New Hippocampal Neurons methods from Spatial Learning Depends on Both the Addition and Removal of New Hippocampal Neurons.
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Learning-Induced Apoptosis Is Critical for Spatial Memory
reagent used in the protocol.
- Use
- We then examined whether learning-induced cell death plays a role in the stabilization of performances in the water maze. To address this issue, at the end of the fourth training session, i.e., when learning-induced cell death becomes apparent, animals were infused in the lateral ventricles with either the pan-caspa...
Intracerebroventricular surgery.
reagent used in the protocol.
- Use
- Guide cannulae were implanted, according to a previously described method [ ], above the rostral ventricle in order not to cause lesions in the hippocampus. Two weeks later, 6 µl (per infusion site) of vehicle (Ringer's solution with 1% DMSO) or of zVAD-fmk (at a concentration of 1 µg of zVAD/µl of ve...
Immunohistochemistry.
reagent used in the protocol.
- Use
- To examine the phenotype of BrdU-IR cells, one in ten sections were incubated with BrdU antibodies (1/500; Accurate), which were revealed using a CY3-anti-rat antibody (1/1,000; Jackson Immunoresearch, http://www.jacksonimmuno.com ). Sections were then incubated with anti-DCX antibodies (1/1,000; Santa Cruz Bi...
Spatial Learning Promotes the Death of Newborn Neurons within a Certain Time Window
These data suggest that learning promotes the death of cells that have reached a certain level of maturation and are older than 5 d. To test this hypothesis, additional groups of animals were trained in the water maze under conditions similar to those of experiment 3, but were injected with BrdU either 3 or 4 d befo...
- Use
- These data suggest that learning promotes the death of cells that have reached a certain level of maturation and are older than 5 d. To test this hypothesis, additional groups of animals were trained in the water maze under conditions similar to those of experiment 3, but were injected with BrdU either 3 or 4 d befo...
Learning-Induced Apoptosis Is Critical for Spatial Memory
We then examined whether learning-induced cell death plays a role in the stabilization of performances in the water maze. To address this issue, at the end of the fourth training session, i.e., when learning-induced cell death becomes apparent, animals were infused in the lateral ventricles with either the pan-caspa...
- Use
- We then examined whether learning-induced cell death plays a role in the stabilization of performances in the water maze. To address this issue, at the end of the fourth training session, i.e., when learning-induced cell death becomes apparent, animals were infused in the lateral ventricles with either the pan-caspa...
Learning-Induced Apoptosis Is Critical for Spatial Memory
To further evaluate the strength of the memory trace after zVAD treatment, a probe test was performed on the seventh day of training. The probe test consisted of exposing animals to the water maze in the absence of the escape platform and recording the time spent in the quadrant of the water maze that contained the...
- Use
- To further evaluate the strength of the memory trace after zVAD treatment, a probe test was performed on the seventh day of training. The probe test consisted of exposing animals to the water maze in the absence of the escape platform and recording the time spent in the quadrant of the water maze that contained the...
Learning-Induced Increases in Apoptosis, Cell Proliferation, and Survival of Newborn Neurons Are Interrelated Processes
It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems then that learning can induce increases in both the survival and death of newborn neurons. For this reason, in a final experiment, we studied t...
- Use
- It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems then that learning can induce increases in both the survival and death of newborn neurons. For this reason, in a final experiment, we studied t...
Materials and Methods
Three-month-old male Sprague-Dawley rats were tested in a water maze according to a previously described method [ ]. Briefly, animals were tested 2 wk following their arrival. The apparatus consisted of a circular swimming pool built of white plastic (180-cm diameter, 60-cm height) filled with water (20 ± 1 ...
- Use
- Three-month-old male Sprague-Dawley rats were tested in a water maze according to a previously described method [ ]. Briefly, animals were tested 2 wk following their arrival. The apparatus consisted of a circular swimming pool built of white plastic (180-cm diameter, 60-cm height) filled with water (20 ± 1 ...
Materials and Methods
In the first experiment ( ), two control groups were used: a Control group (C) consisting of animals that were transferred to the testing room at the same time and with the same procedures as the learning group but that were not exposed to the water maze, and a Yoked group (Y), a control for the stress and motor act...
- Use
- In the first experiment ( ), two control groups were used: a Control group (C) consisting of animals that were transferred to the testing room at the same time and with the same procedures as the learning group but that were not exposed to the water maze, and a Yoked group (Y), a control for the stress and motor act...
Immunohistochemistry.
To examine the phenotype of BrdU-IR cells, one in ten sections were incubated with BrdU antibodies (1/500; Accurate), which were revealed using a CY3-anti-rat antibody (1/1,000; Jackson Immunoresearch, http://www.jacksonimmuno.com ). Sections were then incubated with anti-DCX antibodies (1/1,000; Santa Cruz Bi...
- Use
- To examine the phenotype of BrdU-IR cells, one in ten sections were incubated with BrdU antibodies (1/500; Accurate), which were revealed using a CY3-anti-rat antibody (1/1,000; Jackson Immunoresearch, http://www.jacksonimmuno.com ). Sections were then incubated with anti-DCX antibodies (1/1,000; Santa Cruz Bi...
Electrophysiology.
Hippocampal slices (500 µm) were prepared as described previously [ ] from vehicle- and zVAD -infused rats by an investigator blind to the treatments. Slices were submerged in an oxygenated artificial cerebrospinal fluid (ACSF) comprising (in mM): NaCl 123, KCl 2.5, Na 2 HPO 4 1, NaHCO 3 26.2, Cacl 2 2.4, MgCl...
- Use
- Hippocampal slices (500 µm) were prepared as described previously [ ] from vehicle- and zVAD -infused rats by an investigator blind to the treatments. Slices were submerged in an oxygenated artificial cerebrospinal fluid (ACSF) comprising (in mM): NaCl 123, KCl 2.5, Na 2 HPO 4 1, NaHCO 3 26.2, Cacl 2 2.4, MgCl...
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Learning-Induced Apoptosis Is Critical for Spatial Memory
To further evaluate the strength of the memory trace after zVAD treatment, a probe test was performed on the seventh day of training. The probe test consisted of exposing animals to the water maze in the absence of the escape platform and recording the time spent in the quadrant of the water maze that contained the platform during training (target quadrant). zVAD-infused animals spent less time than vehicle rats in the target quadrant ( B and C, t 22 = 2.01, p ≤ 0.05). In addition, several indices used to measure the efficiency of the swim paths to reach the goal location were also impaired in zVAD-infused animals ( ). These results confirm that the inhibition of apoptosis when animals begin to master the task leads to an impairment of the memory for the platform location.
Learning-Induced Apoptosis Is Critical for Spatial Memory
In order to test for the specificity of the effects of zVAD, we performed several complementary measures and experiments. Thus, after the probe test, animals were tested for their ability to find a visible platform (cued test), which allowed for a measure of visuomotor processes ( ). In this case, zVAD treatment had no measurable effects on behavioral performance. We also evaluated whether zVAD would modify the survival of newly born cells younger than 5 d, because these neurons are normally untouched by learning. This population of cells was labeled by injecting BrdU on day 1 to day 3 of training ( ). Again, zVAD treatment had no measurable effects ( t 22 = 0.87, p = 0.39). Then, in a subsequent experiment (batch 9), we infused zVAD during the first 3 d of training, a period during which learning has no influence on cell death ( B and C). Consequently, if the effects of zVAD are medi...
Learning-Induced Increases in Apoptosis, Cell Proliferation, and Survival of Newborn Neurons Are Interrelated Processes
It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems then that learning can induce increases in both the survival and death of newborn neurons. For this reason, in a final experiment, we studied the relationships between these two phenomena (batch 11). This experiment was performed by injecting the same animals with two thymidine analogs, 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) [ ] ( ). IdU and CldU were injected at different times in order to analyze in the same subject the fate of newborn cells of different ages ( ). IdU was injected 7 d before training in order to label the newborn cells for which survival should be increased by learning. CldU was injected 3 d before the start of the training in order to label newly born cells...
Thymidine analog injections.
Newly born cells were labeled by the incorporation of synthetic thymidine analog (XdU [where X represents Br, Cl, or I]; ). Rats (batches 1, 3-8, and 11) were injected with BrdU (intraperitoneal). The Learning groups received one daily BrdU injection 30 min before the first trials or a single BrdU injection 3 or 4 d before the onset of training. Rats of the 11th experiment received a single injection of IdU and of CldU [ ], respectively, 7 and 3 d before the onset of training, both at equimolar doses of 50 mg BrdU/kg. The control groups were injected with XdU within the same period.
Materials and Methods
Three-month-old male Sprague-Dawley rats were tested in a water maze according to a previously described method [ ]. Briefly, animals were tested 2 wk following their arrival. The apparatus consisted of a circular swimming pool built of white plastic (180-cm diameter, 60-cm height) filled with water (20 ± 1 °C) that has been made opaque by the addition of a nontoxic white cosmetic adjuvant. Before the start of the training, the animals were habituated to the pool for 2 d for 1 min/d. During training, the Learning group (L) was composed of animals that were required to locate the submerged platform, hidden 1.5 cm under the water in a fixed location, using the spatial cues available within the testing room. They were all tested for four trials per day (90 s with an intertrial interval of 30 s and beginning from three different start points that varied randomly each day). If an...
Materials and Methods
In the first experiment ( ), two control groups were used: a Control group (C) consisting of animals that were transferred to the testing room at the same time and with the same procedures as the learning group but that were not exposed to the water maze, and a Yoked group (Y), a control for the stress and motor activity associated with the water-maze training, composed of rats that were placed into the pool without the platform and were paired for the duration of the trial with the Learning animals. In the second experiment, three control groups were used: a Control group, a Yoked group, and an additional group of rats that were trained to find a visible platform (VP) in a fixed location. Animals in this group were all tested for four trials per day (90 s with an intertrial interval of 30 s and beginning from three different start points that varied randomly each day). Because in the...
Intracerebroventricular surgery.
Guide cannulae were implanted, according to a previously described method [ ], above the rostral ventricle in order not to cause lesions in the hippocampus. Two weeks later, 6 µl (per infusion site) of vehicle (Ringer's solution with 1% DMSO) or of zVAD-fmk (at a concentration of 1 µg of zVAD/µl of vehicle; Calbiochem, http://www.emdbiosciences.com/html/CBC/home.html ) [ ] solutions were infused at a constant rate (3 µl/min) in naive or in trained animals immediately after the last trial of the 4th-6th days (batches 8 and 11) or of the 1st-3rd days (batch 9) of training.
Electrophysiology.
Hippocampal slices (500 µm) were prepared as described previously [ ] from vehicle- and zVAD -infused rats by an investigator blind to the treatments. Slices were submerged in an oxygenated artificial cerebrospinal fluid (ACSF) comprising (in mM): NaCl 123, KCl 2.5, Na 2 HPO 4 1, NaHCO 3 26.2, Cacl 2 2.4, MgCl 2 1.2, glucose 10, bicuculline 0.02 (pH 7.4; 295 mosmol.kg -1; room temperature). A concentric bipolar steel electrode was placed in the stratum radiatum to evoke (0.01 Hz) field excitatory postsynaptic potentials (fEPSPs) recorded with a glass electrode filled with ACSF. Data were collected with a multiclamp 700A (Axon Instruments, http://www.axon.com ), filtered at 3 kHz, sampled at 10 kHz and analyzed offline using pClamp 9 (Axon Instruments). The initial slopes of the fEPSPs were measured from approximately 10%-40% of the rising phase. Paired-pulse ratio co...
Measurement outputs
What raw and processed outputs should exist?
(A) Latency to find the escape platform. Learning groups were injected with BrdU either 4 or 3 d before initiation of training as indicated by the syringes. D, day.
- 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 then examined whether learning-induced cell death plays a role in the stabilization of performances in the water maze. To address this issue, at the end of the fourth trainin...
- 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) Latency to reach the hidden platform in animals infused with zVAD ( • ) or vehicle (○). The dashed line represents the mean escape latency across the first four 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
It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems the...
- 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
All data (mean ± standard error of the mean) were analyzed by a Student t -test (two tailed) or by analysis of variance followed by Newman-Keuls test when necessary.
from paperScoring or quantification
Quantify the primary readouts for this experiment: (A) Latency to find the escape platform. Learning groups were injected with BrdU either 4 or 3 d before initiation of training as indicated by the syringes. D, day.; We then examined whether learning-induced cell death plays a role in the stabilization of performances in the water maze. To address this issue, at the end of the fourth trainin...; (A) Latency to reach the hidden platform in animals infused with zVAD ( • ) or vehicle (○). The dashed line represents the mean escape latency across the first four tr...; It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems the....
from paperStatistical comparison
All data (mean ± standard error of the mean) were analyzed by a Student t -test (two tailed) or by analysis of variance followed by Newman-Keuls test when necessary. Correl...; In order to characterize the relationship between learning and changes in cell death, we correlated the performance in the water maze (mean latency to find the platform after 3...
from paperReporting output
Report representative outputs alongside summary comparisons for (A) Latency to find the escape platform. Learning groups were injected with BrdU either 4 or 3 d before initiation of training as indicated by the syringes. D, day., We then examined whether learning-induced cell death plays a role in the stabilization of performances in the water maze. To address this issue, at the end of the fourth trainin..., (A) Latency to reach the hidden platform in animals infused with zVAD ( • ) or vehicle (○). The dashed line represents the mean escape latency across the first four tr..., It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems the....
inferred from protocolStructured statistical methods
All data (mean ± standard error of the mean) were analyzed by a Student t -test (two tailed) or by analysis of variance followed by Newman-Keuls test when necessary. Correl...; In order to characterize the relationship between learning and changes in cell death, we correlated the performance in the water maze (mean latency to find the platform after 3...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
To further evaluate the strength of the memory trace after zVAD treatment, a probe test was performed on the seventh day of training. The probe test consisted of exposing animals to the water maze in the absence of the escape platform and recording the time spent in the quadrant of the water maze that contained the platform during training (target quadrant). zVAD-infused animals spent less time than vehicle rats in the target quadrant ( B and C, t 22 = 2.01, p ≤ 0.05). In addition, several indices used to measure the efficiency of the swim paths to reach the goal location were also impaired in zVAD-infused animals ( ). These results confirm that the inhibition of apoptosis when animals begin to master the task leads to an impairment of the memory for the platform location.
In order to test for the specificity of the effects of zVAD, we performed several complementary measures and experiments. Thus, after the probe test, animals were tested for their ability to find a visible platform (cued test), which allowed for a measure of visuomotor processes ( ). In this case, zVAD treatment had no measurable effects on behavioral performance. We also evaluated whether zVAD would modify the survival of newly born cells younger than 5 d, because these neurons are normally untouched by learning. This population of cells was labeled by injecting BrdU on day 1 to day 3 of training ( ). Again, zVAD treatment had no measurable effects ( t 22 = 0.87, p = 0.39). Then, in a subsequent experiment (batch 9), we infused zVAD during the first 3 d of training, a period during which learning has no influence on cell death ( B and C). Consequently, if the effects of zVAD are mediated by a blockade of learning-induced cell death, this schedule of treatment should have no behavioral effects. Indeed, we found that under these conditions, zVAD infusions did not impair spatial memory ( ). Finally, we performed physiological recordings in the Ammon's horn, a part of the hippocamp...
It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems then that learning can induce increases in both the survival and death of newborn neurons. For this reason, in a final experiment, we studied the relationships between these two phenomena (batch 11). This experiment was performed by injecting the same animals with two thymidine analogs, 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) [ ] ( ). IdU and CldU were injected at different times in order to analyze in the same subject the fate of newborn cells of different ages ( ). IdU was injected 7 d before training in order to label the newborn cells for which survival should be increased by learning. CldU was injected 3 d before the start of the training in order to label newly born cells that should die as a consequence of learning. Animals in these experiments were also infused either with vehicle or zVAD at the end of the fourth through sixth days of training. As found in the previous experiment, vehicle- and zVAD-infused animals did not differ in their latency to find the hidden...
Newly born cells were labeled by the incorporation of synthetic thymidine analog (XdU [where X represents Br, Cl, or I]; ). Rats (batches 1, 3-8, and 11) were injected with BrdU (intraperitoneal). The Learning groups received one daily BrdU injection 30 min before the first trials or a single BrdU injection 3 or 4 d before the onset of training. Rats of the 11th experiment received a single injection of IdU and of CldU [ ], respectively, 7 and 3 d before the onset of training, both at equimolar doses of 50 mg BrdU/kg. The control groups were injected with XdU within the same period.
Three-month-old male Sprague-Dawley rats were tested in a water maze according to a previously described method [ ]. Briefly, animals were tested 2 wk following their arrival. The apparatus consisted of a circular swimming pool built of white plastic (180-cm diameter, 60-cm height) filled with water (20 ± 1 °C) that has been made opaque by the addition of a nontoxic white cosmetic adjuvant. Before the start of the training, the animals were habituated to the pool for 2 d for 1 min/d. During training, the Learning group (L) was composed of animals that were required to locate the submerged platform, hidden 1.5 cm under the water in a fixed location, using the spatial cues available within the testing room. They were all tested for four trials per day (90 s with an intertrial interval of 30 s and beginning from three different start points that varied randomly each day). If an animal did not find the platform, it was set on it at the end of the trial. The time to reach the platform (latency in seconds) was collected using a video camera fixed to the ceiling of the room and connected to a computerized tracking system (Videotrack; Viewpoint, http://www.viewpoint.fr/en_EU/...
In the first experiment ( ), two control groups were used: a Control group (C) consisting of animals that were transferred to the testing room at the same time and with the same procedures as the learning group but that were not exposed to the water maze, and a Yoked group (Y), a control for the stress and motor activity associated with the water-maze training, composed of rats that were placed into the pool without the platform and were paired for the duration of the trial with the Learning animals. In the second experiment, three control groups were used: a Control group, a Yoked group, and an additional group of rats that were trained to find a visible platform (VP) in a fixed location. Animals in this group were all tested for four trials per day (90 s with an intertrial interval of 30 s and beginning from three different start points that varied randomly each day). Because in the first experiment the Yoked and Control groups did not differ for cell genesis or cell death, and because in the second experiment the Visual Platform, Yoked, and Control groups also did not differ, only the Control group was used for subsequent experiments. All experiments were performed in accorda...
Guide cannulae were implanted, according to a previously described method [ ], above the rostral ventricle in order not to cause lesions in the hippocampus. Two weeks later, 6 µl (per infusion site) of vehicle (Ringer's solution with 1% DMSO) or of zVAD-fmk (at a concentration of 1 µg of zVAD/µl of vehicle; Calbiochem, http://www.emdbiosciences.com/html/CBC/home.html ) [ ] solutions were infused at a constant rate (3 µl/min) in naive or in trained animals immediately after the last trial of the 4th-6th days (batches 8 and 11) or of the 1st-3rd days (batch 9) of training.
Hippocampal slices (500 µm) were prepared as described previously [ ] from vehicle- and zVAD -infused rats by an investigator blind to the treatments. Slices were submerged in an oxygenated artificial cerebrospinal fluid (ACSF) comprising (in mM): NaCl 123, KCl 2.5, Na 2 HPO 4 1, NaHCO 3 26.2, Cacl 2 2.4, MgCl 2 1.2, glucose 10, bicuculline 0.02 (pH 7.4; 295 mosmol.kg -1; room temperature). A concentric bipolar steel electrode was placed in the stratum radiatum to evoke (0.01 Hz) field excitatory postsynaptic potentials (fEPSPs) recorded with a glass electrode filled with ACSF. Data were collected with a multiclamp 700A (Axon Instruments, http://www.axon.com ), filtered at 3 kHz, sampled at 10 kHz and analyzed offline using pClamp 9 (Axon Instruments). The initial slopes of the fEPSPs were measured from approximately 10%-40% of the rising phase. Paired-pulse ratio corresponds to the slope ratio of the second fEPSP to the first fEPSP.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Spatial Learning Depends on Both the Addition and Removal of New Hippocampal Neurons methods",
"description": "Evidence-backed execution summary for Spatial Learning Depends on Both the Addition and Removal of New Hippocampal Neurons methods from Spatial Learning Depends on Both the Addition and Removal of New Hippocampal Neurons.",
"totalTime": "PT31M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Learning-Induced Apoptosis Is Critical for Spatial Memory",
"text": "To further evaluate the strength of the memory trace after zVAD treatment, a probe test was performed on the seventh day of training. The probe test consisted of exposing animals to the water maze in the absence of the escape platform and recording the time spent in the quadrant of the water maze that contained the platform during training (target quadrant). zVAD-infused animals spent less time than vehicle rats in the target quadrant ( B and C, t 22 = 2.01, p ≤ 0.05). In addition, several indices used to measure the efficiency of the swim paths to reach the goal location were also impaired in zVAD-infused animals ( ). These results confirm that the inhibition of apoptosis when animals begin to master the task leads to an impairment of the memory for the platform location."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Learning-Induced Apoptosis Is Critical for Spatial Memory",
"text": "In order to test for the specificity of the effects of zVAD, we performed several complementary measures and experiments. Thus, after the probe test, animals were tested for their ability to find a visible platform (cued test), which allowed for a measure of visuomotor processes ( ). In this case, zVAD treatment had no measurable effects on behavioral performance. We also evaluated whether zVAD would modify the survival of newly born cells younger than 5 d, because these neurons are normally untouched by learning. This population of cells was labeled by injecting BrdU on day 1 to day 3 of training ( ). Again, zVAD treatment had no measurable effects ( t 22 = 0.87, p = 0.39). Then, in a subsequent experiment (batch 9), we infused zVAD during the first 3 d of training, a period during which learning has no influence on cell death ( B and C). Consequently, if the effects of zVAD are medi..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Learning-Induced Increases in Apoptosis, Cell Proliferation, and Survival of Newborn Neurons Are Interrelated Processes",
"text": "It has previously been shown that training in a water maze increases the survival of newborn neurons that were produced 1 wk before the start of the training [, ]. It seems then that learning can induce increases in both the survival and death of newborn neurons. For this reason, in a final experiment, we studied the relationships between these two phenomena (batch 11). This experiment was performed by injecting the same animals with two thymidine analogs, 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) [ ] ( ). IdU and CldU were injected at different times in order to analyze in the same subject the fate of newborn cells of different ages ( ). IdU was injected 7 d before training in order to label the newborn cells for which survival should be increased by learning. CldU was injected 3 d before the start of the training in order to label newly born cells..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Thymidine analog injections.",
"text": "Newly born cells were labeled by the incorporation of synthetic thymidine analog (XdU [where X represents Br, Cl, or I]; ). Rats (batches 1, 3-8, and 11) were injected with BrdU (intraperitoneal). The Learning groups received one daily BrdU injection 30 min before the first trials or a single BrdU injection 3 or 4 d before the onset of training. Rats of the 11th experiment received a single injection of IdU and of CldU [ ], respectively, 7 and 3 d before the onset of training, both at equimolar doses of 50 mg BrdU/kg. The control groups were injected with XdU within the same period."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Materials and Methods",
"text": "Three-month-old male Sprague-Dawley rats were tested in a water maze according to a previously described method [ ]. Briefly, animals were tested 2 wk following their arrival. The apparatus consisted of a circular swimming pool built of white plastic (180-cm diameter, 60-cm height) filled with water (20 ± 1 °C) that has been made opaque by the addition of a nontoxic white cosmetic adjuvant. Before the start of the training, the animals were habituated to the pool for 2 d for 1 min/d. During training, the Learning group (L) was composed of animals that were required to locate the submerged platform, hidden 1.5 cm under the water in a fixed location, using the spatial cues available within the testing room. They were all tested for four trials per day (90 s with an intertrial interval of 30 s and beginning from three different start points that varied randomly each day). If an..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Materials and Methods",
"text": "In the first experiment ( ), two control groups were used: a Control group (C) consisting of animals that were transferred to the testing room at the same time and with the same procedures as the learning group but that were not exposed to the water maze, and a Yoked group (Y), a control for the stress and motor activity associated with the water-maze training, composed of rats that were placed into the pool without the platform and were paired for the duration of the trial with the Learning animals. In the second experiment, three control groups were used: a Control group, a Yoked group, and an additional group of rats that were trained to find a visible platform (VP) in a fixed location. Animals in this group were all tested for four trials per day (90 s with an intertrial interval of 30 s and beginning from three different start points that varied randomly each day). Because in the..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Intracerebroventricular surgery.",
"text": "Guide cannulae were implanted, according to a previously described method [ ], above the rostral ventricle in order not to cause lesions in the hippocampus. Two weeks later, 6 µl (per infusion site) of vehicle (Ringer's solution with 1% DMSO) or of zVAD-fmk (at a concentration of 1 µg of zVAD/µl of vehicle; Calbiochem, http://www.emdbiosciences.com/html/CBC/home.html ) [ ] solutions were infused at a constant rate (3 µl/min) in naive or in trained animals immediately after the last trial of the 4th-6th days (batches 8 and 11) or of the 1st-3rd days (batch 9) of training."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Electrophysiology.",
"text": "Hippocampal slices (500 µm) were prepared as described previously [ ] from vehicle- and zVAD -infused rats by an investigator blind to the treatments. Slices were submerged in an oxygenated artificial cerebrospinal fluid (ACSF) comprising (in mM): NaCl 123, KCl 2.5, Na 2 HPO 4 1, NaHCO 3 26.2, Cacl 2 2.4, MgCl 2 1.2, glucose 10, bicuculline 0.02 (pH 7.4; 295 mosmol.kg -1; room temperature). A concentric bipolar steel electrode was placed in the stratum radiatum to evoke (0.01 Hz) field excitatory postsynaptic potentials (fEPSPs) recorded with a glass electrode filled with ACSF. Data were collected with a multiclamp 700A (Axon Instruments, http://www.axon.com ), filtered at 3 kHz, sampled at 10 kHz and analyzed offline using pClamp 9 (Axon Instruments). The initial slopes of the fEPSPs were measured from approximately 10%-40% of the rising phase. Paired-pulse ratio co..."
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