Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
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?
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
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource 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
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods",
"description": "Protocol for Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods",
"step": [
{
"@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."
},
{
"@type": "HowToStep",
"position": 3,
"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."
},
{
"@type": "HowToStep",
"position": 4,
"name": "FTY20 (long term).",
"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",
"position": 6,
"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",
"position": 7,
"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...."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Regulation of learning and memory by meningeal immunity: a key role for IL-4",
"datePublished": "2010",
"author": [
{
"@type": "Person",
"name": "Noël C. Derecki"
},
{
"@type": "Person",
"name": "Amber N. Cardani"
},
{
"@type": "Person",
"name": "Chun Hui Yang"
},
{
"@type": "Person",
"name": "Kayla M. Quinnies"
},
{
"@type": "Person",
"name": "Anastasia Crihfield"
},
{
"@type": "Person",
"name": "Kevin R. Lynch"
},
{
"@type": "Person",
"name": "Jonathan Kipnis"
}
],
"identifier": "10.1084/jem.20091419"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Regulation of learning and memory by meningeal immunity: a key role for IL-4 methods",
"item": "https://replicatescience.com/experiments/regulation-of-learning-and-memory-by-meningeal-immunity-a-key-role-for-il-4-methods-no-235-l-c-derecki-pmc2867291/regulation-of-learning-and-memory-by-meningeal-immunity-a-key-role-for-il-4-mlpgwbgl"
}
]
}
]