Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus methods
Aim. Evidence-backed execution summary for Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus methods from Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus.
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?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- Three days after litter birth, dams and their respective offspring were housed together in the vapor chamber apparatus. Litters were randomly assigned to either alcohol treatment or control. Starting at 10:00 a.m., litters were exposed to alcohol via vapor inhalation for 4 h daily during their light cycle, from...
Reverse transcriptase polymerase chain reaction
reagent used in the protocol.
- Use
- Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. ). Additionally, to address the duration of effects, samples were collected at P11-13. Eight pups (from a total of 8 litters) were used...
Immunohistochemistry
reagent used in the protocol.
- Use
- On P4, P6, and P45 (Fig. ), 4 pups (from a total of 4 litters) per treatment group were anesthetized by injection of ketamine (250 mg/kg). Animals were transcardially perfused first with phosphate buffered saline (PBS), pH 7.4, containing procaine hydrochloride (1 g/L) and heparin (1 USP unit/L) for...
Immunohistochemistry
reagent used in the protocol.
- Use
- All immunohistochemical analyses were performed by a blinded researcher using ImageJ (NIH, Bethesda, MD). At least two sections per animal, at least five sections apart, were averaged for each antibody. Sections were imaged on a Nikon TE2000 microscope (Nikon, Melville, NY) with a nuance spectral camera (Quorum, mod...
PAE does not induce apoptosis in the cerebellar vermis during the first withdrawal period at P4
reagent used in the protocol.
- Use
- Since caspase 3 has been shown to have non-apoptotic roles in the cerebellum during development, we also assessed for apoptosis by TUNEL assay on P4 (Additional file ). We found no difference between air control and PAE animals in the EGL (Additional file c, two-way ANOVA, interaction F( 2,17 ) = 0.13, p...
PAE-induced increases in astrocytic GFAP expression occur with or without microglial morphological transitions: possi...
reagent used in the protocol.
- Use
- Astrocyte activation varies heavily based on the type of insult and neighboring environmental cues (reviewed by [ ]). As such, if glial cells are playing a role in PAE, it is likely that astrocytes in the hippocampus and cerebellar vermis have distinct functions. In some cases, astrocytes can be heavily anti-inflamm...
Additional files
reagent used in the protocol.
- Use
- Postnatal alcohol exposure (PAE) does not increase TUNEL staining in the cerebellar vermis. Effect of PAE on TUNEL staining in the cerebellar vermis during the first withdrawal period on P4. Animals were treated as described in Fig.. Representative images of the cerebellar vermis were stained with a TUNEL ass...
Materials and methods
Three days after litter birth, dams and their respective offspring were housed together in the vapor chamber apparatus. Litters were randomly assigned to either alcohol treatment or control. Starting at 10:00 a.m., litters were exposed to alcohol via vapor inhalation for 4 h daily during their light cycle, from...
- Use
- Three days after litter birth, dams and their respective offspring were housed together in the vapor chamber apparatus. Litters were randomly assigned to either alcohol treatment or control. Starting at 10:00 a.m., litters were exposed to alcohol via vapor inhalation for 4 h daily during their light cycle, from...
Materials and methods
Nursing ability was assessed by counting the number of pups in each litter who had a visible presence of milk in their stomachs on the mornings after each exposure day. Finally, maternal care was assessed as previously described [ ]. Briefly, on P4, starting at exposure hour 0, litters were filmed for 15 min ou...
- Use
- Nursing ability was assessed by counting the number of pups in each litter who had a visible presence of milk in their stomachs on the mornings after each exposure day. Finally, maternal care was assessed as previously described [ ]. Briefly, on P4, starting at exposure hour 0, litters were filmed for 15 min ou...
Immunohistochemistry
All immunohistochemical analyses were performed by a blinded researcher using ImageJ (NIH, Bethesda, MD). At least two sections per animal, at least five sections apart, were averaged for each antibody. Sections were imaged on a Nikon TE2000 microscope (Nikon, Melville, NY) with a nuance spectral camera (Quorum, mod...
- Use
- All immunohistochemical analyses were performed by a blinded researcher using ImageJ (NIH, Bethesda, MD). At least two sections per animal, at least five sections apart, were averaged for each antibody. Sections were imaged on a Nikon TE2000 microscope (Nikon, Melville, NY) with a nuance spectral camera (Quorum, mod...
Immunohistochemistry
To quantify neuronal number, the thickness of the cell layer and/or the total cell number was assessed. In the case of cell layer thickness, measurements were made randomly in three separate locations and averaged per image. To quantify neuronal number, cells were counted using unbiased stereological techniques, bas...
- Use
- To quantify neuronal number, the thickness of the cell layer and/or the total cell number was assessed. In the case of cell layer thickness, measurements were made randomly in three separate locations and averaged per image. To quantify neuronal number, cells were counted using unbiased stereological techniques, bas...
Gait analysis
Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-...
- Use
- Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-...
Contextual fear conditioning
Multiple pre-exposure contextual fear conditioning was used to test hippocampal-dependent behavioral differences. Pre-exposing animals to the context 1 day prior to shock conditioning, which has been shown to increase freezing, is impaired in a model of neonatal alcohol exposure [ ]. Animals aged P36-48 (...
- Use
- Multiple pre-exposure contextual fear conditioning was used to test hippocampal-dependent behavioral differences. Pre-exposing animals to the context 1 day prior to shock conditioning, which has been shown to increase freezing, is impaired in a model of neonatal alcohol exposure [ ]. Animals aged P36-48 (...
Statistical analysis
All statistics were analyzed with Prism Version 6.05 (GraphPad Software, San Diego, CA). Data were analyzed by student's unpaired t test or two-way ANOVA followed by Bonferroni post hoc test. Significance was determined as p < 0.05. Data are shown as mean ± SEM.
- Use
- All statistics were analyzed with Prism Version 6.05 (GraphPad Software, San Diego, CA). Data were analyzed by student's unpaired t test or two-way ANOVA followed by Bonferroni post hoc test. Significance was determined as p < 0.05. Data are shown as mean ± SEM.
PAE-induced increases in astrocytic GFAP expression occur with or without microglial morphological transitions: possi...
As stated above, neuronal loss was seen with this paradigm in the cerebellar vermis but not in the hippocampus. Interestingly, an increase in amoeboid microglia occurred only in the cerebellar vermis, while robust elevations in astrocytic GFAP expression was observed in both brain regions. While microglial morpholog...
- Use
- As stated above, neuronal loss was seen with this paradigm in the cerebellar vermis but not in the hippocampus. Interestingly, an increase in amoeboid microglia occurred only in the cerebellar vermis, while robust elevations in astrocytic GFAP expression was observed in both brain regions. While microglial morpholog...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All statistics were analyzed with Prism Version 6.05 (GraphPad Software, San Diego, CA). Data were analyzed by student's unpaired t test or two-way ANOVA followed by Bonferroni post hoc test. Significance was determined as p < 0.05. Data are shown as mean ± SEM.
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
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?
Reverse transcriptase polymerase chain reaction
Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. ). Additionally, to address the duration of effects, samples were collected at P11-13. Eight pups (from a total of 8 litters) were used per treatment group. Animals were anesthetized with ketamine (250 mg/kg). Whole hippocampi and cerebellar vermises were collected. Tissue was homogenized by sonication in ice cold RNeasy Lysis Buffer and RNA was extracted using an RNeasy Mini Kit according to the manufacturer's instructions (SABiosciences/Qiagen, Valencia, CA). The RNA was stored at -20 °C until use. cDNA was synthesized from 1 µg of RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat# 4368813, Foster City, CA) according to the manufacturer&...
Immunohistochemistry
On P4, P6, and P45 (Fig. ), 4 pups (from a total of 4 litters) per treatment group were anesthetized by injection of ketamine (250 mg/kg). Animals were transcardially perfused first with phosphate buffered saline (PBS), pH 7.4, containing procaine hydrochloride (1 g/L) and heparin (1 USP unit/L) for 4 min, followed by ice cold 4 % paraformaldehyde in PBS. Brains were removed by decapitation and placed in 4 % paraformaldehyde for 48 h at 4 °C, then 30 % sucrose for 24-48 h at 4 °C. Brains were embedded in Optimal Cutting Temperature compound (Fisher Healthcare, Houston, TX) and frozen before sectioning on a cryostat (Microm, model# HM 505E, Walldorf, Germany) at 16 µm. Sections for staining were incubated with PBS containing 1 % bovine serum albumin, 0.2 % Triton X-100, and 5 % of either do...
Gait analysis
Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-HSC; two to three animals per litter were used from a total of 4 litters per treatment group. Rats aged P45-50 (Fig. ) were placed on an enclosed glass walkway illuminated from above by a red fluorescent light and along the walkway by green light-emitting diode lights. Disruption of the green light allowed for tracking of paw prints, while disruption of red light allowed for visualization of silhouettes. Rats were allowed to walk freely across the runway to a dark box, containing bedding from their home cage, located at the opposite end. A digital high-speed cam...
Measurement outputs
What raw and processed outputs should exist?
Three days after litter birth, dams and their respective offspring were housed together in the vapor chamber apparatus. Litters were randomly assigned to either alcohol treatmen...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Nursing ability was assessed by counting the number of pups in each litter who had a visible presence of milk in their stomachs on the mornings after each exposure day. Finally,...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. )....
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To quantify neuronal number, the thickness of the cell layer and/or the total cell number was assessed. In the case of cell layer thickness, measurements were made randomly in t...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
All statistics were analyzed with Prism Version 6.05 (GraphPad Software, San Diego, CA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Three days after litter birth, dams and their respective offspring were housed together in the vapor chamber apparatus. Litters were randomly assigned to either alcohol treatmen...; Nursing ability was assessed by counting the number of pups in each litter who had a visible presence of milk in their stomachs on the mornings after each exposure day. Finally,...; Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. )....; To quantify neuronal number, the thickness of the cell layer and/or the total cell number was assessed. In the case of cell layer thickness, measurements were made randomly in t....
from paperStatistical comparison
All statistics were analyzed with Prism Version 6.05 (GraphPad Software, San Diego, CA). Data were analyzed by student's unpaired t test or two-way ANOVA followed by Bonfe...; On the morning after the first exposure (i.e., first withdrawal period at P4; as shown in Fig. ), no effect of PAE was observed on Purkinje cell number across any of the l...; Similarly, on P45, there was no significant change in either neuronal number (Fig., two-way ANOVA, interaction F( 2,18 ) = 0.43, p = 0.655; regio...; On P45, the number of Purkinje cells continued to be decreased in lobule regions I-III and IX-X, but not in region IV-VII (Fig., two-way ANOVA, interact...
from paperReporting output
Report representative outputs alongside summary comparisons for Three days after litter birth, dams and their respective offspring were housed together in the vapor chamber apparatus. Litters were randomly assigned to either alcohol treatmen..., Nursing ability was assessed by counting the number of pups in each litter who had a visible presence of milk in their stomachs on the mornings after each exposure day. Finally,..., Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. )...., To quantify neuronal number, the thickness of the cell layer and/or the total cell number was assessed. In the case of cell layer thickness, measurements were made randomly in t....
inferred from protocolStructured statistical methods
All statistics were analyzed with Prism Version 6.05 (GraphPad Software, San Diego, CA). Data were analyzed by student's unpaired t test or two-way ANOVA followed by Bonfe...; On the morning after the first exposure (i.e., first withdrawal period at P4; as shown in Fig. ), no effect of PAE was observed on Purkinje cell number across any of the l...; Similarly, on P45, there was no significant change in either neuronal number (Fig., two-way ANOVA, interaction F( 2,18 ) = 0.43, p = 0.655; regio...; On P45, the number of Purkinje cells continued to be decreased in lobule regions I-III and IX-X, but not in region IV-VII (Fig., two-way ANOVA, interact...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. ). Additionally, to address the duration of effects, samples were collected at P11-13. Eight pups (from a total of 8 litters) were used per treatment group. Animals were anesthetized with ketamine (250 mg/kg). Whole hippocampi and cerebellar vermises were collected. Tissue was homogenized by sonication in ice cold RNeasy Lysis Buffer and RNA was extracted using an RNeasy Mini Kit according to the manufacturer's instructions (SABiosciences/Qiagen, Valencia, CA). The RNA was stored at -20 °C until use. cDNA was synthesized from 1 µg of RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat# 4368813, Foster City, CA) according to the manufacturer's instructions. The following primers from SABiosciences/Qiagen were used: interleukin-1β (IL-1β, cat# PPR06480B), tumor necrosis factor α (TNFα, cat# PPR06411F), interleukin-10 (IL10, cat# PPR06479A), transforming growth factor β (TGFβ, cat# PPR06430B), and hypox...
On P4, P6, and P45 (Fig. ), 4 pups (from a total of 4 litters) per treatment group were anesthetized by injection of ketamine (250 mg/kg). Animals were transcardially perfused first with phosphate buffered saline (PBS), pH 7.4, containing procaine hydrochloride (1 g/L) and heparin (1 USP unit/L) for 4 min, followed by ice cold 4 % paraformaldehyde in PBS. Brains were removed by decapitation and placed in 4 % paraformaldehyde for 48 h at 4 °C, then 30 % sucrose for 24-48 h at 4 °C. Brains were embedded in Optimal Cutting Temperature compound (Fisher Healthcare, Houston, TX) and frozen before sectioning on a cryostat (Microm, model# HM 505E, Walldorf, Germany) at 16 µm. Sections for staining were incubated with PBS containing 1 % bovine serum albumin, 0.2 % Triton X-100, and 5 % of either donkey or goat serum to match the host species of the secondary antibodies described below. Primary antibodies were applied overnight at 4 °C. Astrocytes were stained with rabbit anti-glial fibrillary acidic protein (GFAP, 1:500, Dako, cat# 019-19741, Carpinteria, CA), microglia with rabbit...
Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-HSC; two to three animals per litter were used from a total of 4 litters per treatment group. Rats aged P45-50 (Fig. ) were placed on an enclosed glass walkway illuminated from above by a red fluorescent light and along the walkway by green light-emitting diode lights. Disruption of the green light allowed for tracking of paw prints, while disruption of red light allowed for visualization of silhouettes. Rats were allowed to walk freely across the runway to a dark box, containing bedding from their home cage, located at the opposite end. A digital high-speed camera recorded each trial until three compliant trials were captured in which the animal did not stop or turn around and crossed the walkway within 0.5-10 s with a maximum body speed variation of less than 60 %. PAE significantly increased the total number of trials required to achieve...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus methods",
"description": "Evidence-backed execution summary for Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus methods from Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus.",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Reverse transcriptase polymerase chain reaction",
"text": "Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. ). Additionally, to address the duration of effects, samples were collected at P11-13. Eight pups (from a total of 8 litters) were used per treatment group. Animals were anesthetized with ketamine (250 mg/kg). Whole hippocampi and cerebellar vermises were collected. Tissue was homogenized by sonication in ice cold RNeasy Lysis Buffer and RNA was extracted using an RNeasy Mini Kit according to the manufacturer's instructions (SABiosciences/Qiagen, Valencia, CA). The RNA was stored at -20 °C until use. cDNA was synthesized from 1 µg of RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat# 4368813, Foster City, CA) according to the manufacturer&..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Immunohistochemistry",
"text": "On P4, P6, and P45 (Fig. ), 4 pups (from a total of 4 litters) per treatment group were anesthetized by injection of ketamine (250 mg/kg). Animals were transcardially perfused first with phosphate buffered saline (PBS), pH 7.4, containing procaine hydrochloride (1 g/L) and heparin (1 USP unit/L) for 4 min, followed by ice cold 4 % paraformaldehyde in PBS. Brains were removed by decapitation and placed in 4 % paraformaldehyde for 48 h at 4 °C, then 30 % sucrose for 24-48 h at 4 °C. Brains were embedded in Optimal Cutting Temperature compound (Fisher Healthcare, Houston, TX) and frozen before sectioning on a cryostat (Microm, model# HM 505E, Walldorf, Germany) at 16 µm. Sections for staining were incubated with PBS containing 1 % bovine serum albumin, 0.2 % Triton X-100, and 5 % of either do..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Gait analysis",
"text": "Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-HSC; two to three animals per litter were used from a total of 4 litters per treatment group. Rats aged P45-50 (Fig. ) were placed on an enclosed glass walkway illuminated from above by a red fluorescent light and along the walkway by green light-emitting diode lights. Disruption of the green light allowed for tracking of paw prints, while disruption of red light allowed for visualization of silhouettes. Rats were allowed to walk freely across the runway to a dark box, containing bedding from their home cage, located at the opposite end. A digital high-speed cam..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Materials and methods"
},
{
"@type": "HowToTool",
"name": "Materials and methods"
},
{
"@type": "HowToTool",
"name": "Immunohistochemistry"
},
{
"@type": "HowToTool",
"name": "Immunohistochemistry"
},
{
"@type": "HowToTool",
"name": "Gait analysis"
},
{
"@type": "HowToTool",
"name": "Contextual fear conditioning"
},
{
"@type": "HowToTool",
"name": "Statistical analysis"
},
{
"@type": "HowToTool",
"name": "PAE-induced increases in astrocytic GFAP expression occur with or without microglial morphological transitions: possi..."
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Materials and methods"
},
{
"@type": "HowToSupply",
"name": "Reverse transcriptase polymerase chain reaction"
},
{
"@type": "HowToSupply",
"name": "Immunohistochemistry"
},
{
"@type": "HowToSupply",
"name": "Immunohistochemistry"
},
{
"@type": "HowToSupply",
"name": "PAE does not induce apoptosis in the cerebellar vermis during the first withdrawal period at P4"
},
{
"@type": "HowToSupply",
"name": "PAE-induced increases in astrocytic GFAP expression occur with or without microglial morphological transitions: possi..."
},
{
"@type": "HowToSupply",
"name": "Additional files"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus",
"datePublished": "2015",
"author": [
{
"@type": "Person",
"name": "Lauren A. Topper"
},
{
"@type": "Person",
"name": "Brian C. Baculis"
},
{
"@type": "Person",
"name": "C. Fernando Valenzuela"
}
],
"identifier": "10.1186/s12974-015-0382-9)"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus methods",
"item": "https://replicatescience.com/experiments/exposure-of-neonatal-rats-to-alcohol-has-differential-effects-on-neuroinflammation-and-neuronal-survival-in-the-cerebellum-and-hippocampus-methods-lauren-a-topper-pmc4558631/exposure-of-neonatal-rats-to-alcohol-has-differential-effects-on-neuroinflammation-and-neuronal-surv-mlph92cu"
}
]
}
]