Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods
Aim. Evidence-backed execution summary for Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods from Regulation of learning and memory by meningeal immunity: a key role for IL-4.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Meningeal T cell response after MWM learning and memory task performance
reagent used in the protocol.
- Use
- We examined meningeal immunity after treatment with FTY720 or anti-VLA4. With FTY720 treatment, CD4 + T cells in the meninges were dramatically reduced in numbers ( ). The presented populations were gated for CD45 + and CD3 + and then examined for CD4 expression. Although a massive general reduction in all T cells w...
Meningeal myeloid cell response after cognitive task performance and its T cell-dependent regulation
reagent used in the protocol.
- Use
- To address the possibility that T cells might be involved in the regulation of the phenotype of meningeal myeloid cells, we examined their expression of proinflammatory cytokines upon acute depletion of meningeal T cells using either FTY720 or anti-VLA4 antibody treatment. CD11b + cells isolated from the meninges of...
The essential role of IL-4 in the regulation of meningeal myeloid cells and in cognitive task performance
reagent used in the protocol.
- Use
- To gain insight into the possible downstream neural mechanism mediated by the meningeal cytokine milieu, we examined the effect of different cytokines and their combinations on astrocytic expression of BDNF. BDNF has been shown to be a key molecule in several learning and memory paradigms, including the MWM (; ). I...
Drug treatments
reagent used in the protocol.
- Use
- A rat monoclonal antibody to mouse VLA4 (clone PS/2) was affinity purified from hybridoma supernatants and used with the permission of K. Ley (La Jolla Institute of Allergy and Immunology, San Diego, CA). Animals were given three separate injections i.p. (1.2 mg/mouse in 250 µl of 0.1 M PBS) of antibody (or an...
T cell isolation and transfer
reagent used in the protocol.
- Use
- Lymph nodes (axillary, inguinal, superficial, and deep cervical) were harvested, mashed, and passed twice through 70-µm nylon screens. T cells were purified (enriched by negative selection) using autoMACS, a Pan T Cell Isolation Kit, and the Possel-S program (all from Miltenyi Biotec), using the negative fracti...
Fear conditioning
reagent used in the protocol.
- Use
- Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects' behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spa...
Bone marrow isolation
reagent used in the protocol.
- Use
- Mice were sacrificed using CO 2 and saturated with 70% alcohol. Skin was removed from the lower part of the body. Tissue was removed from hind legs with scissors and dissected away from the body. Remaining tissue was cleaned from the tibial and femoral bones and bones were separated at the knee joint. Bone ends were...
FACS of meningeal isolates
reagent used in the protocol.
- Use
- Mice were thoroughly transcardially perfused with 0.1 M PBS, pH 7.4, for 5 min immediately after the last training trial. Heads were removed and skulls were quickly stripped of all flesh. Mandibles were next removed, as was all skull material rostral to maxillae. Surgical scissors (Fine Science Tools) were used to r...
Meningeal T cell response after MWM learning and memory task performance
In an attempt to better understand the cellular and molecular mechanisms underlying the observed beneficial effect of T cells on performance in the MWM, we examined T cell response after task performance. Immunohistological examination of brain tissue did not reveal any T cells in brain parenchyma from either naive...
- Use
- In an attempt to better understand the cellular and molecular mechanisms underlying the observed beneficial effect of T cells on performance in the MWM, we examined T cell response after task performance. Immunohistological examination of brain tissue did not reveal any T cells in brain parenchyma from either naive...
Meningeal T cell response after MWM learning and memory task performance
Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid spaces occupied by CD3 + cells are presented, and arrowheads point to CD3 + cells. Bar, 50 µm (a). CD3 + cells were stereologically count...
- Use
- Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid spaces occupied by CD3 + cells are presented, and arrowheads point to CD3 + cells. Bar, 50 µm (a). CD3 + cells were stereologically count...
Meningeal myeloid cell response after cognitive task performance and its T cell-dependent regulation
T cell-dependent regulation of meningeal myeloid cell phenotype in MWM-trained mice. (a) Whole brain cryosections were double labeled for CD68 and MHCII. Positive cells (myeloid) in the choroid plexus of the third ventricle are presented. Bars, 50 µm. (b-e) FACS analyses were performed at least thre...
- Use
- T cell-dependent regulation of meningeal myeloid cell phenotype in MWM-trained mice. (a) Whole brain cryosections were double labeled for CD68 and MHCII. Positive cells (myeloid) in the choroid plexus of the third ventricle are presented. Bars, 50 µm. (b-e) FACS analyses were performed at least thre...
The essential role of IL-4 in the regulation of meningeal myeloid cells and in cognitive task performance
T cell-derived IL-4 regulates meningeal myeloid cell phenotype and influences learning and memory. All behavior experiments were performed by an experimenter blinded to the identity of experimental groups and were recorded with the EthoVision video tracking system. Representative experiments are shown out of a...
- Use
- T cell-derived IL-4 regulates meningeal myeloid cell phenotype and influences learning and memory. All behavior experiments were performed by an experimenter blinded to the identity of experimental groups and were recorded with the EthoVision video tracking system. Representative experiments are shown out of a...
The essential role of IL-4 in the regulation of meningeal myeloid cells and in cognitive task performance
To link the cognitive impairment seen in IL-4 -/- mice with meningeal immunity, we examined meningeal myeloid cells for cytokine expression. A significant increase in proinflammatory cytokine production (TNF) by meningeal myeloid cells from IL-4 -/- mice was evident as compared with myeloid c...
- Use
- To link the cognitive impairment seen in IL-4 -/- mice with meningeal immunity, we examined meningeal myeloid cells for cytokine expression. A significant increase in proinflammatory cytokine production (TNF) by meningeal myeloid cells from IL-4 -/- mice was evident as compared with myeloid c...
T cell isolation and transfer
Lymph nodes (axillary, inguinal, superficial, and deep cervical) were harvested, mashed, and passed twice through 70-µm nylon screens. T cells were purified (enriched by negative selection) using autoMACS, a Pan T Cell Isolation Kit, and the Possel-S program (all from Miltenyi Biotec), using the negative fracti...
- Use
- Lymph nodes (axillary, inguinal, superficial, and deep cervical) were harvested, mashed, and passed twice through 70-µm nylon screens. T cells were purified (enriched by negative selection) using autoMACS, a Pan T Cell Isolation Kit, and the Possel-S program (all from Miltenyi Biotec), using the negative fracti...
MWM
Mice were given four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter. The water temperature was kept between 21 and 22°C. Water was made opaque with nontoxic tempera. Within the testing room, only distal visual shape...
- Use
- Mice were given four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter. The water temperature was kept between 21 and 22°C. Water was made opaque with nontoxic tempera. Within the testing room, only distal visual shape...
Fear conditioning
Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects' behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spa...
- Use
- Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects' behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spa...
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MATERIALS AND METHODS
Inbred male adult (8-10-wk-old) BALB/c/BySmn, CBySmn.CB17- Prkdc scid /J, B6.129P2-IL4 tm1Cgn /J, C57BL/6-Tg(UBC-GFP)30Scha/J, and C57BL/6J mice were purchased from the Jackson Laboratory. All animals were housed in temperature- and humidity-controlled rooms, maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.), and age matched in each experiment. All strains were kept in identical housing conditions. The lifespan of SCID and IL-4 -/- mice is comparable to that of wild-type mice, and there are no specific dietary or housing requirements for either mutant strain. Animal protocols were approved by the University of Virginia Institutional Animal Care and Use Committee. All procedures complied with regulations of the Institutional Animal Care and Use Committee at the University of Virginia.
Drug treatments
A rat monoclonal antibody to mouse VLA4 (clone PS/2) was affinity purified from hybridoma supernatants and used with the permission of K. Ley (La Jolla Institute of Allergy and Immunology, San Diego, CA). Animals were given three separate injections i.p. (1.2 mg/mouse in 250 µl of 0.1 M PBS) of antibody (or an equivalent amount of rat IgG for control mice). The first dose was given 5 d before the beginning of MWM training, the second dose was given on the day before the beginning of the task, and the last injection was given after 4 d of training.
FTY720 (short term).
Animals were treated daily with an oral administration (1 mg/kg in 0.1M PBS by gavage) of FTY720 (or an equivalent amount of PBS) starting 1 wk before the initiation of training in the MWM spatial learning and memory task. Animals were continued on daily oral FTY720 treatment throughout training.
FTY20 (long term).
FTY720 was dissolved in drinking water (4.3 × 10 -3 mg/ml); bottles were changed daily for 8 wk before and during MWM training.
MWM
Mice were given four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter. The water temperature was kept between 21 and 22°C. Water was made opaque with nontoxic tempera. Within the testing room, only distal visual shape and object cues were available to the mice to aid in location of the submerged platform. The BALB/c mice used in this study have poor vision and cannot fully see the shapes and objects, although they can distinguish light from darkness. In previous studies we have addressed this possible problem by using lights as visual cues, and for the visible trial we attached a glowing stick to the platform. Changing the objects on the walls to lights and adding the glowing stick significantly improves the learning ability of BALB/c mice in this task. The escape latency, i.e., the tim...
Fear conditioning
Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects' behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spaced 1.5 cm apart (center to center). The rods were wired to a shock generator and scrambler for the delivery of footshock. The chambers were cleaned with a 70% ethanol solution, and pans containing a thin film of the same solution were placed underneath the grid floors. Background noise (60 dB, A scale) was supplied by a fan positioned adjacent to the boxes. The mice were placed in the conditioning context for 2 min before receiving five tone (30 s, 2.8 kHz, 85 dB)/shock (2 s, 0.5 mA) pairings spaced by 1-min inter-trial intervals. 48 h later the mice received a 5-min test...
Irradiation and bone marrow replenishment
Adult wild-type C57BL/6J mice were subjected to a lethal split dose of γ irradiation (350 rad followed 48 h later by 950 rad). 3 h after the second irradiation, mice were injected with 4 × 10 6 bone marrow cells freshly isolated from identical wild-type mice or from IL-4 -/- mice. After irradiation, mice were kept on drinking water fortified with sulfamethoxazole for 3 wk to limit infection.
FACS of meningeal isolates
Mice were thoroughly transcardially perfused with 0.1 M PBS, pH 7.4, for 5 min immediately after the last training trial. Heads were removed and skulls were quickly stripped of all flesh. Mandibles were next removed, as was all skull material rostral to maxillae. Surgical scissors (Fine Science Tools) were used to remove skull tops, cutting clockwise, beginning and ending inferior to the right posttympanic hook. Brains and superior skulls were immediately placed in ice-cold FACS buffer (0.1 M PBS, 1 mM EDTA, 1% BSA, pH 7.4). Meninges (dura, arachnoid, and pia mater) were carefully removed from the interior aspect of skulls and surfaces of brains with forceps (Dumont #5; Fine Science Tools). Meninges from each group ( n = 4) were pooled. Meningeal tissue was gently pressed through 70-µm nylon mesh cell strainers with sterile plastic plungers (BD) to yield a single-cell suspension....
Measurement outputs
What raw and processed outputs should exist?
Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid sp...
- 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
To further characterize T cells from naive and MWM-trained wild-type mice, we examined their activation phenotype. A substantial increase in CD69-expressing meningeal CD4 + T ce...
- 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 of the aforementioned meningeal myeloid cells was based on a series of gates applied to all samples, as indicated in, using a viability dye (live/dead gate for the eli...
- 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 examined meningeal immunity after treatment with FTY720 or anti-VLA4. With FTY720 treatment, CD4 + T cells in the meninges were dramatically reduced in numbers ( ). The prese...
- 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
In an attempt to better understand the cellular and molecular mechanisms underlying the observed beneficial effect of T cells on performance in the MWM, we examined T cell response after task performance.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid sp...; To further characterize T cells from naive and MWM-trained wild-type mice, we examined their activation phenotype. A substantial increase in CD69-expressing meningeal CD4 + T ce...; Analysis of the aforementioned meningeal myeloid cells was based on a series of gates applied to all samples, as indicated in, using a viability dye (live/dead gate for the eli...; We examined meningeal immunity after treatment with FTY720 or anti-VLA4. With FTY720 treatment, CD4 + T cells in the meninges were dramatically reduced in numbers ( ). The prese....
from paperStatistical comparison
In an attempt to better understand the cellular and molecular mechanisms underlying the observed beneficial effect of T cells on performance in the MWM, we examined T cell respo...; Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid sp...; T cell-derived IL-4 regulates meningeal myeloid cell phenotype and influences learning and memory. All behavior experiments were performed by an experimenter blinded to th...; To link the cognitive impairment seen in IL-4 -/- mice with meningeal immunity, we examined meningeal myeloid cells for cytokine expression. A significant increase i...
from paperReporting output
Report representative outputs alongside summary comparisons for Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid sp..., To further characterize T cells from naive and MWM-trained wild-type mice, we examined their activation phenotype. A substantial increase in CD69-expressing meningeal CD4 + T ce..., Analysis of the aforementioned meningeal myeloid cells was based on a series of gates applied to all samples, as indicated in, using a viability dye (live/dead gate for the eli..., We examined meningeal immunity after treatment with FTY720 or anti-VLA4. With FTY720 treatment, CD4 + T cells in the meninges were dramatically reduced in numbers ( ). The prese....
inferred from protocolStructured statistical methods
In an attempt to better understand the cellular and molecular mechanisms underlying the observed beneficial effect of T cells on performance in the MWM, we examined T cell respo...; Accumulation and activation of IL-4-producing T cells in the meningeal spaces of MWM-trained mice. (a and b) Whole brain cryosections were labeled for CD3. Subarachnoid sp...; T cell-derived IL-4 regulates meningeal myeloid cell phenotype and influences learning and memory. All behavior experiments were performed by an experimenter blinded to th...; To link the cognitive impairment seen in IL-4 -/- mice with meningeal immunity, we examined meningeal myeloid cells for cytokine expression. A significant increase i...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Inbred male adult (8-10-wk-old) BALB/c/BySmn, CBySmn.CB17- Prkdc scid /J, B6.129P2-IL4 tm1Cgn /J, C57BL/6-Tg(UBC-GFP)30Scha/J, and C57BL/6J mice were purchased from the Jackson Laboratory. All animals were housed in temperature- and humidity-controlled rooms, maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.), and age matched in each experiment. All strains were kept in identical housing conditions. The lifespan of SCID and IL-4 -/- mice is comparable to that of wild-type mice, and there are no specific dietary or housing requirements for either mutant strain. Animal protocols were approved by the University of Virginia Institutional Animal Care and Use Committee. All procedures complied with regulations of the Institutional Animal Care and Use Committee at the University of Virginia.
A rat monoclonal antibody to mouse VLA4 (clone PS/2) was affinity purified from hybridoma supernatants and used with the permission of K. Ley (La Jolla Institute of Allergy and Immunology, San Diego, CA). Animals were given three separate injections i.p. (1.2 mg/mouse in 250 µl of 0.1 M PBS) of antibody (or an equivalent amount of rat IgG for control mice). The first dose was given 5 d before the beginning of MWM training, the second dose was given on the day before the beginning of the task, and the last injection was given after 4 d of training.
Animals were treated daily with an oral administration (1 mg/kg in 0.1M PBS by gavage) of FTY720 (or an equivalent amount of PBS) starting 1 wk before the initiation of training in the MWM spatial learning and memory task. Animals were continued on daily oral FTY720 treatment throughout training.
FTY720 was dissolved in drinking water (4.3 × 10 -3 mg/ml); bottles were changed daily for 8 wk before and during MWM training.
Mice were given four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter. The water temperature was kept between 21 and 22°C. Water was made opaque with nontoxic tempera. Within the testing room, only distal visual shape and object cues were available to the mice to aid in location of the submerged platform. The BALB/c mice used in this study have poor vision and cannot fully see the shapes and objects, although they can distinguish light from darkness. In previous studies we have addressed this possible problem by using lights as visual cues, and for the visible trial we attached a glowing stick to the platform. Changing the objects on the walls to lights and adding the glowing stick significantly improves the learning ability of BALB/c mice in this task. The escape latency, i.e., the time required by the mouse to find and climb onto the platform, was recorded for up to 60 s. Each mouse was allowed to remain on the platform for 20 s and was then moved from the maze to its home cage. If the mouse did not find the platform within 60 s, it was manually placed on the platform and return...
Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects' behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spaced 1.5 cm apart (center to center). The rods were wired to a shock generator and scrambler for the delivery of footshock. The chambers were cleaned with a 70% ethanol solution, and pans containing a thin film of the same solution were placed underneath the grid floors. Background noise (60 dB, A scale) was supplied by a fan positioned adjacent to the boxes. The mice were placed in the conditioning context for 2 min before receiving five tone (30 s, 2.8 kHz, 85 dB)/shock (2 s, 0.5 mA) pairings spaced by 1-min inter-trial intervals. 48 h later the mice received a 5-min test in the training context. Freezing was scored by an automated system during all sessions and used as an index of memory. All fear conditioning or testing was performed between 10 a.m. and 3 p.m. during the lights-on phase. The experimenter was blind to the genotype or drug status of all animals durin...
Adult wild-type C57BL/6J mice were subjected to a lethal split dose of γ irradiation (350 rad followed 48 h later by 950 rad). 3 h after the second irradiation, mice were injected with 4 × 10 6 bone marrow cells freshly isolated from identical wild-type mice or from IL-4 -/- mice. After irradiation, mice were kept on drinking water fortified with sulfamethoxazole for 3 wk to limit infection.
Mice were thoroughly transcardially perfused with 0.1 M PBS, pH 7.4, for 5 min immediately after the last training trial. Heads were removed and skulls were quickly stripped of all flesh. Mandibles were next removed, as was all skull material rostral to maxillae. Surgical scissors (Fine Science Tools) were used to remove skull tops, cutting clockwise, beginning and ending inferior to the right posttympanic hook. Brains and superior skulls were immediately placed in ice-cold FACS buffer (0.1 M PBS, 1 mM EDTA, 1% BSA, pH 7.4). Meninges (dura, arachnoid, and pia mater) were carefully removed from the interior aspect of skulls and surfaces of brains with forceps (Dumont #5; Fine Science Tools). Meninges from each group ( n = 4) were pooled. Meningeal tissue was gently pressed through 70-µm nylon mesh cell strainers with sterile plastic plungers (BD) to yield a single-cell suspension. Cells were centrifuged at 1,100 RPM at 4°C for 10 min, the supernatant was removed, and cells were resuspended in ice-cold FACS buffer. Cells were stained for extracellular markers with antibodies to CD11b conjugated to FITC or PE; CD45 conjugated to allophycocyanin (APC), APC-Cy7, or efluor 4...
Machine-readable layer
[
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"name": "Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods",
"description": "Evidence-backed execution summary for Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods from Regulation of learning and memory by meningeal immunity: a key role for IL-4.",
"totalTime": "PT12M",
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{
"@type": "HowToStep",
"position": 1,
"name": "MATERIALS AND METHODS",
"text": "Inbred male adult (8-10-wk-old) BALB/c/BySmn, CBySmn.CB17- Prkdc scid /J, B6.129P2-IL4 tm1Cgn /J, C57BL/6-Tg(UBC-GFP)30Scha/J, and C57BL/6J mice were purchased from the Jackson Laboratory. All animals were housed in temperature- and humidity-controlled rooms, maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.), and age matched in each experiment. All strains were kept in identical housing conditions. The lifespan of SCID and IL-4 -/- mice is comparable to that of wild-type mice, and there are no specific dietary or housing requirements for either mutant strain. Animal protocols were approved by the University of Virginia Institutional Animal Care and Use Committee. All procedures complied with regulations of the Institutional Animal Care and Use Committee at the University of Virginia."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Drug treatments",
"text": "A rat monoclonal antibody to mouse VLA4 (clone PS/2) was affinity purified from hybridoma supernatants and used with the permission of K. Ley (La Jolla Institute of Allergy and Immunology, San Diego, CA). Animals were given three separate injections i.p. (1.2 mg/mouse in 250 µl of 0.1 M PBS) of antibody (or an equivalent amount of rat IgG for control mice). The first dose was given 5 d before the beginning of MWM training, the second dose was given on the day before the beginning of the task, and the last injection was given after 4 d of training."
},
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"@type": "HowToStep",
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"name": "FTY720 (short term).",
"text": "Animals were treated daily with an oral administration (1 mg/kg in 0.1M PBS by gavage) of FTY720 (or an equivalent amount of PBS) starting 1 wk before the initiation of training in the MWM spatial learning and memory task. Animals were continued on daily oral FTY720 treatment throughout training."
},
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"@type": "HowToStep",
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"text": "FTY720 was dissolved in drinking water (4.3 × 10 -3 mg/ml); bottles were changed daily for 8 wk before and during MWM training."
},
{
"@type": "HowToStep",
"position": 5,
"name": "MWM",
"text": "Mice were given four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter. The water temperature was kept between 21 and 22°C. Water was made opaque with nontoxic tempera. Within the testing room, only distal visual shape and object cues were available to the mice to aid in location of the submerged platform. The BALB/c mice used in this study have poor vision and cannot fully see the shapes and objects, although they can distinguish light from darkness. In previous studies we have addressed this possible problem by using lights as visual cues, and for the visible trial we attached a glowing stick to the platform. Changing the objects on the walls to lights and adding the glowing stick significantly improves the learning ability of BALB/c mice in this task. The escape latency, i.e., the tim..."
},
{
"@type": "HowToStep",
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"name": "Fear conditioning",
"text": "Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects' behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spaced 1.5 cm apart (center to center). The rods were wired to a shock generator and scrambler for the delivery of footshock. The chambers were cleaned with a 70% ethanol solution, and pans containing a thin film of the same solution were placed underneath the grid floors. Background noise (60 dB, A scale) was supplied by a fan positioned adjacent to the boxes. The mice were placed in the conditioning context for 2 min before receiving five tone (30 s, 2.8 kHz, 85 dB)/shock (2 s, 0.5 mA) pairings spaced by 1-min inter-trial intervals. 48 h later the mice received a 5-min test..."
},
{
"@type": "HowToStep",
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"name": "Irradiation and bone marrow replenishment",
"text": "Adult wild-type C57BL/6J mice were subjected to a lethal split dose of γ irradiation (350 rad followed 48 h later by 950 rad). 3 h after the second irradiation, mice were injected with 4 × 10 6 bone marrow cells freshly isolated from identical wild-type mice or from IL-4 -/- mice. After irradiation, mice were kept on drinking water fortified with sulfamethoxazole for 3 wk to limit infection."
},
{
"@type": "HowToStep",
"position": 8,
"name": "FACS of meningeal isolates",
"text": "Mice were thoroughly transcardially perfused with 0.1 M PBS, pH 7.4, for 5 min immediately after the last training trial. Heads were removed and skulls were quickly stripped of all flesh. Mandibles were next removed, as was all skull material rostral to maxillae. Surgical scissors (Fine Science Tools) were used to remove skull tops, cutting clockwise, beginning and ending inferior to the right posttympanic hook. Brains and superior skulls were immediately placed in ice-cold FACS buffer (0.1 M PBS, 1 mM EDTA, 1% BSA, pH 7.4). Meninges (dura, arachnoid, and pia mater) were carefully removed from the interior aspect of skulls and surfaces of brains with forceps (Dumont #5; Fine Science Tools). Meninges from each group ( n = 4) were pooled. Meningeal tissue was gently pressed through 70-µm nylon mesh cell strainers with sterile plastic plungers (BD) to yield a single-cell suspension...."
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