Cognitive neurobehavior and synaptic plasticity after vagus nerve stimulation in female rats: Interaction between estrous cycle and neurostimulation 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?
Methods
In compliance with all federal regulations governing the protection of animals and research and DoD Instruction 3216.01, the Wright-Patterson Air Force Base IACUC approved this study, and all activities were conducted in an AAALAC International accredited facility. Female Sprague-Dawley rats (Charles River) were grouped housed on a 12 h light cycle (lights on at 6 am) with ad libitum access to food and water. After at least a week of acclimation, rats were surgically implanted with VN electrode cuffs. Starting immediately after surgery, rats were single housed for the duration of the study. After at least 10 days of recovery from surgery, animals were assigned to either SHAM or VNS groups based on their cessation of breathing outcome. One subset of rats underwent behavioral testing paired with a single session of VNS ( n = 18-24 per group). Outlined...
Cuff Implantation and VNS
As previously described (Olsen et al. ), animals underwent surgical VNS electrode implantation around the left cervical VN prior to stimulation and behavioral testing. Briefly, electrode wires (90% Pt/10% Ir, 0.0011″ from Sigmund Cohn, Mt Vernon, New York) were soldered to gold pins connected to an Omnetics 4-channel head cap. Flamed ends of the electrode wires (~ 2-3 mm) were strung through catheter tubing (0.08″ outer diameter, 0.04″ inner diameter). Impedance of the electrode wires was checked prior to surgical implantation (2-4.5 kΩ range). While under anesthesia (isoflurane- 5% induction, 2-3% maintenance), the Omnetics 4-channel head cap was secured to the skull of the rat using stainless steel screws and dental cement. The electrode wires were subcutaneously tunneled from the skull to the bottom of the left neck. The left cerv...
Novel Object Recognition (NOR)
The NOR test was used to detect changes in recognition memory as previously described [6]. Panasonic cameras and Ethovision v11.5 (Noldus, Leesburg, VA) were used to measure rodent behavior in NOR. Rats were exposed to two similar objects (~ 8 cm radius, ~ 6 cm tall crown shaped glass item spray painted black) for three min. After 24 h, rats were exposed to the same object and a novel object (~ 8 cm radius, ~ 10 cm tall rook shaped glass item spray painted black). Objects were placed on opposing sides of the arenas 47 cm apart. Rats were also exposed to the Elevated Zero Maze paradigm as previously described (17) for five min prior to the beginning of NOR object exposure on testing day only. On training day and testing day, rats habituated to the arena (60 cm × 60 cm × 38...
Passive Avoidance Task (PAT)
The PAT paradigm was used to measure associative aversion learning (Gemini Avoidance System, San Diego Instruments Inc., San Diego, CA) as previously described (Olsen et al. ). Rats were habituated to the apparatus for five min. The next day, rats were placed into a well-lit box (25 cm × 21 cm × 17 cm) and allowed to move into a dark box of the same size. If the rat chose to cross over to the dark box, a one-time 0.75 mA foot shock (FS) was administered followed by an additional 30 s in the dark box to facilitate aversive training by associating the dark box with the FS. After 24 h the rat was returned to the well-lit box. Latency to enter the dark box from the well-lit box was measured and the number of rats 'hesitant' (> 160 s) to enter the dark box versus 'not hesitant' to enter the d...
Elevated Zero Maze (EZM)
The EZM test was used to identify changes in anxiolytic-like behavior. Panasonic cameras and Ethovision v11.5 (Noldus, Leesburg, VA) were used to measure rodent behavior in EZM. The 100 cm diameter circular platform was elevated 65 cm above the ground floor. Divided into four equal arms, two opposing arms were open and the other two opposing arms were enclosed with 30 cm dark walls. The open arm regions were surrounded by a 1 cm edge. Animals were placed in the middle of a closed arm (randomized for each trial between the relative left or right closed arm). The animal was allowed to explore without an experimenter within view for five minutes while their activity was monitored using Ethovision XT (v11.5, Noldus).
IHC Staining
Rats were saline perfused, followed by brain extraction and fixation in 4% paraformaldehyde for 48 h and 30% sucrose for at least three days. Fixed brain tissue was sliced using a microtome and 30 µm coronal hippocampal slices (1:6 serial section staining) were collected for immunostaining of BDNF protein. Tissue was blocked (PBS + 2% goat serum, 0.2% Triton X-100) for 1 h at room temperature and incubated in rabbit anti-BDNF (1:250, sc-20981, Santa Cruz) overnight at 4 °C. The next day the tissue was incubated in mouse anti-NeuN (1:2500, MAB377, Sigma) for 2 h at room temperature. After all primary antibody incubations were complete, hippocampal slices were incubated in fluorescent secondary (Alexa Fluor 488 and 594, 111-545-144 and 115-585-146, Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The tissue...
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 (6)
In compliance with all federal regulations governing the protection of animals and research and DoD Instruction 3216.01, the Wright-Patterson Air Force Base IACUC approved this study, and all activities were conducted in an AAALAC International accredited facility. Female Sprague-Dawley rats (Charles River) were grouped housed on a 12 h light cycle (lights on at 6 am) with ad libitum access to food and water. After at least a week of acclimation, rats were surgically implanted with VN electrode cuffs. Starting immediately after surgery, rats were single housed for the duration of the study. After at least 10 days of recovery from surgery, animals were assigned to either SHAM or VNS groups based on their cessation of breathing outcome. One subset of rats underwent behavioral testing paired with a single session of VNS ( n = 18-24 per group). Outlined in Fig. A, all behavior animals underwent all neurobehavior testing paradigms. All behavior animals were euthanized at the end of the experiment, which was 48 h after VNS administration for post-mortem tissue collection for hippocampal IHC analysis. Ex vivo electrophysiological recordings...
As previously described (Olsen et al. ), animals underwent surgical VNS electrode implantation around the left cervical VN prior to stimulation and behavioral testing. Briefly, electrode wires (90% Pt/10% Ir, 0.0011″ from Sigmund Cohn, Mt Vernon, New York) were soldered to gold pins connected to an Omnetics 4-channel head cap. Flamed ends of the electrode wires (~ 2-3 mm) were strung through catheter tubing (0.08″ outer diameter, 0.04″ inner diameter). Impedance of the electrode wires was checked prior to surgical implantation (2-4.5 kΩ range). While under anesthesia (isoflurane- 5% induction, 2-3% maintenance), the Omnetics 4-channel head cap was secured to the skull of the rat using stainless steel screws and dental cement. The electrode wires were subcutaneously tunneled from the skull to the bottom of the left neck. The left cervical VN was separated from the carotid artery and unsheathed using a glass nerve hook. As previously described, direct contact between the exposed electrodes and the VN was confirmed during surgery by conducting the cessation of breathing test. Briefly, the cessation of breathing test was conducted...
The NOR test was used to detect changes in recognition memory as previously described [6]. Panasonic cameras and Ethovision v11.5 (Noldus, Leesburg, VA) were used to measure rodent behavior in NOR. Rats were exposed to two similar objects (~ 8 cm radius, ~ 6 cm tall crown shaped glass item spray painted black) for three min. After 24 h, rats were exposed to the same object and a novel object (~ 8 cm radius, ~ 10 cm tall rook shaped glass item spray painted black). Objects were placed on opposing sides of the arenas 47 cm apart. Rats were also exposed to the Elevated Zero Maze paradigm as previously described (17) for five min prior to the beginning of NOR object exposure on testing day only. On training day and testing day, rats habituated to the arena (60 cm × 60 cm × 38 cm) for two min prior to object exposure for three min. Object exploration activity was monitored using Ethovision XT (v11.5). Novel object preference (NP) on testing day was calculated using the following equation, with N_novel defined as the amount of time spent exploring the novel object within...
The PAT paradigm was used to measure associative aversion learning (Gemini Avoidance System, San Diego Instruments Inc., San Diego, CA) as previously described (Olsen et al. ). Rats were habituated to the apparatus for five min. The next day, rats were placed into a well-lit box (25 cm × 21 cm × 17 cm) and allowed to move into a dark box of the same size. If the rat chose to cross over to the dark box, a one-time 0.75 mA foot shock (FS) was administered followed by an additional 30 s in the dark box to facilitate aversive training by associating the dark box with the FS. After 24 h the rat was returned to the well-lit box. Latency to enter the dark box from the well-lit box was measured and the number of rats 'hesitant' (> 160 s) to enter the dark box versus 'not hesitant' to enter the dark box was analyzed to determine retention of associative learning.
The EZM test was used to identify changes in anxiolytic-like behavior. Panasonic cameras and Ethovision v11.5 (Noldus, Leesburg, VA) were used to measure rodent behavior in EZM. The 100 cm diameter circular platform was elevated 65 cm above the ground floor. Divided into four equal arms, two opposing arms were open and the other two opposing arms were enclosed with 30 cm dark walls. The open arm regions were surrounded by a 1 cm edge. Animals were placed in the middle of a closed arm (randomized for each trial between the relative left or right closed arm). The animal was allowed to explore without an experimenter within view for five minutes while their activity was monitored using Ethovision XT (v11.5, Noldus).
Rats were saline perfused, followed by brain extraction and fixation in 4% paraformaldehyde for 48 h and 30% sucrose for at least three days. Fixed brain tissue was sliced using a microtome and 30 µm coronal hippocampal slices (1:6 serial section staining) were collected for immunostaining of BDNF protein. Tissue was blocked (PBS + 2% goat serum, 0.2% Triton X-100) for 1 h at room temperature and incubated in rabbit anti-BDNF (1:250, sc-20981, Santa Cruz) overnight at 4 °C. The next day the tissue was incubated in mouse anti-NeuN (1:2500, MAB377, Sigma) for 2 h at room temperature. After all primary antibody incubations were complete, hippocampal slices were incubated in fluorescent secondary (Alexa Fluor 488 and 594, 111-545-144 and 115-585-146, Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The tissue was mounted and coverslipped using Fluoroshield with dapi (F6057, Sigma). Fluorescent images were collected using an Olympus microscope and quantified using Image J and QuPath. The pyramidal layer of the CA1/CA2/CA3 and subgranular zone of the dentate gyrus (DG) were identified using NeuN. Quantifi...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Cognitive neurobehavior and synaptic plasticity after vagus nerve stimulation in female rats: Interaction between estrous cycle and neurostimulation methods",
"description": "Protocol for Cognitive neurobehavior and synaptic plasticity after vagus nerve stimulation in female rats: Interaction between estrous cycle and neurostimulation methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Methods",
"text": "In compliance with all federal regulations governing the protection of animals and research and DoD Instruction 3216.01, the Wright-Patterson Air Force Base IACUC approved this study, and all activities were conducted in an AAALAC International accredited facility. Female Sprague-Dawley rats (Charles River) were grouped housed on a 12 h light cycle (lights on at 6 am) with ad libitum access to food and water. After at least a week of acclimation, rats were surgically implanted with VN electrode cuffs. Starting immediately after surgery, rats were single housed for the duration of the study. After at least 10 days of recovery from surgery, animals were assigned to either SHAM or VNS groups based on their cessation of breathing outcome. One subset of rats underwent behavioral testing paired with a single session of VNS ( n = 18-24 per group). Outlined..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Cuff Implantation and VNS",
"text": "As previously described (Olsen et al. ), animals underwent surgical VNS electrode implantation around the left cervical VN prior to stimulation and behavioral testing. Briefly, electrode wires (90% Pt/10% Ir, 0.0011″ from Sigmund Cohn, Mt Vernon, New York) were soldered to gold pins connected to an Omnetics 4-channel head cap. Flamed ends of the electrode wires (~ 2-3 mm) were strung through catheter tubing (0.08″ outer diameter, 0.04″ inner diameter). Impedance of the electrode wires was checked prior to surgical implantation (2-4.5 kΩ range). While under anesthesia (isoflurane- 5% induction, 2-3% maintenance), the Omnetics 4-channel head cap was secured to the skull of the rat using stainless steel screws and dental cement. The electrode wires were subcutaneously tunneled from the skull to the bottom of the left neck. The left cerv..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Novel Object Recognition (NOR)",
"text": "The NOR test was used to detect changes in recognition memory as previously described [6]. Panasonic cameras and Ethovision v11.5 (Noldus, Leesburg, VA) were used to measure rodent behavior in NOR. Rats were exposed to two similar objects (~ 8 cm radius, ~ 6 cm tall crown shaped glass item spray painted black) for three min. After 24 h, rats were exposed to the same object and a novel object (~ 8 cm radius, ~ 10 cm tall rook shaped glass item spray painted black). Objects were placed on opposing sides of the arenas 47 cm apart. Rats were also exposed to the Elevated Zero Maze paradigm as previously described (17) for five min prior to the beginning of NOR object exposure on testing day only. On training day and testing day, rats habituated to the arena (60 cm × 60 cm × 38..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Passive Avoidance Task (PAT)",
"text": "The PAT paradigm was used to measure associative aversion learning (Gemini Avoidance System, San Diego Instruments Inc., San Diego, CA) as previously described (Olsen et al. ). Rats were habituated to the apparatus for five min. The next day, rats were placed into a well-lit box (25 cm × 21 cm × 17 cm) and allowed to move into a dark box of the same size. If the rat chose to cross over to the dark box, a one-time 0.75 mA foot shock (FS) was administered followed by an additional 30 s in the dark box to facilitate aversive training by associating the dark box with the FS. After 24 h the rat was returned to the well-lit box. Latency to enter the dark box from the well-lit box was measured and the number of rats 'hesitant' (> 160 s) to enter the dark box versus 'not hesitant' to enter the d..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Elevated Zero Maze (EZM)",
"text": "The EZM test was used to identify changes in anxiolytic-like behavior. Panasonic cameras and Ethovision v11.5 (Noldus, Leesburg, VA) were used to measure rodent behavior in EZM. The 100 cm diameter circular platform was elevated 65 cm above the ground floor. Divided into four equal arms, two opposing arms were open and the other two opposing arms were enclosed with 30 cm dark walls. The open arm regions were surrounded by a 1 cm edge. Animals were placed in the middle of a closed arm (randomized for each trial between the relative left or right closed arm). The animal was allowed to explore without an experimenter within view for five minutes while their activity was monitored using Ethovision XT (v11.5, Noldus)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "IHC Staining",
"text": "Rats were saline perfused, followed by brain extraction and fixation in 4% paraformaldehyde for 48 h and 30% sucrose for at least three days. Fixed brain tissue was sliced using a microtome and 30 µm coronal hippocampal slices (1:6 serial section staining) were collected for immunostaining of BDNF protein. Tissue was blocked (PBS + 2% goat serum, 0.2% Triton X-100) for 1 h at room temperature and incubated in rabbit anti-BDNF (1:250, sc-20981, Santa Cruz) overnight at 4 °C. The next day the tissue was incubated in mouse anti-NeuN (1:2500, MAB377, Sigma) for 2 h at room temperature. After all primary antibody incubations were complete, hippocampal slices were incubated in fluorescent secondary (Alexa Fluor 488 and 594, 111-545-144 and 115-585-146, Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The tissue..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Cognitive neurobehavior and synaptic plasticity after vagus nerve stimulation in female rats: Interaction between estrous cycle and neurostimulation",
"datePublished": "2026",
"author": [
{
"@type": "Person",
"name": "Laura K. Olsen"
},
{
"@type": "Person",
"name": "Krysten A. Jones"
},
{
"@type": "Person",
"name": "Birendra Sharma"
},
{
"@type": "Person",
"name": "Victoria T. Ethridge"
},
{
"@type": "Person",
"name": "Nathan M. Gargas"
},
{
"@type": "Person",
"name": "Sylvia D. Cunningham"
},
{
"@type": "Person",
"name": "Joyce G. Rohan"
},
{
"@type": "Person",
"name": "Candice N. Hatcher-Solis"
}
],
"identifier": "10.1186/s42234-025-00196-3"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Cognitive neurobehavior and synaptic plasticity after vagus nerve stimulation in female rats: Interaction between estrous cycle and neurostimulation methods",
"item": "https://replicatescience.com/experiments/cognitive-neurobehavior-and-synaptic-plasticity-after-vagus-nerve-stimulation-in-female-rats-interaction-between-estrous-cycle-and-neurostimulation-methods-laura-k-olsen-pmc1286291/cognitive-neurobehavior-and-synaptic-plasticity-after-vagus-nerve-stimulation-in"
}
]
}
]