Parental olfactory experience influences behavior and neural structure in subsequent generations methods
Aim. Evidence-backed execution summary for Parental olfactory experience influences behavior and neural structure in subsequent generations methods from Parental olfactory experience influences behavior and neural structure in subsequent generations.
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mouse
Subject model for the experiment.
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Transgenerational olfactory sensitivity after F0 conditioning
reagent used in the protocol.
- Use
- Studies that have examined the effect of parental stress after conception, either in utero or postnatally, have often found an anxiogenic phenotype in the offspring,. Using an elevated plus maze to assay for anxiety-like behavior, we found that prior, rather limited foot shock conditioning of the F0 generation, di...
Transgenerational olfactory sensitivity after F0 conditioning
reagent used in the protocol.
- Use
- Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue. In our case, to assay for behavioral sensitivity to an odor, we used a modified FPS protocol that consists of odor presentation before the s...
Transgenerational olfactory sensitivity after F0 conditioning
reagent used in the protocol.
- Use
- To further corroborate the enhanced behavioral sensitivity to the F0-conditioned odor, we conducted an independent behavioral assay that directly probes behavioral sensitivity using an odor concentration curve and the association time of the mice with these concentrations. We found that F1-Ace males were able to det...
Inheritance of effects in the F2 and IVF-derived generations
reagent used in the protocol.
- Use
- When tested in our behavioral assay, F2-Ace-C57 mice exposed to odors for the first time showed increased OPS to acetophenone compared with F2-Prop-C57 mice, whereas F2-Prop-C57 mice showed an increased OPS to propanol ( ). This persistent behavioral sensitivity to the F0-conditioned odor was accompanied by correspo...
Altered epigenetic signature at Olfr151 (M71) locus in sperm
reagent used in the protocol.
- Use
- Published data also support the idea of epigenetic marking in sperm by indicating that sperm-associated histones are retained with chromatin of the paternal genome at the one-cell embryo stage,. To investigate the possibility that histone modifications mark the Olfr151 (M71) locus, we collected sperm from F0-Ace a...
IVF
reagent used in the protocol.
- Use
- IVF was carried out by the Emory Transgenic Mouse Facility (TMF) located in a different building from our colony ( http://med.emory.edu/research/core_labs/transgenic_mouse/ ) across the Emory Campus. Briefly, F0-M71 males were fear conditioned either to acetophenone or propanol as outlined above. 10 d after fear con...
β-galactosidase staining, quantitation of MOE OSN number and glomerular area in bulb
reagent used in the protocol.
- Use
- The MOE and olfactory bulbs of 2-month-old M71-LacZ mice were processed for β-galactosidase staining, and then M71 OSN number and glomerular area were quantitated using previously published protocols. Briefly, lateral whole-mount MOE and brains were rapidly dissected and placed into 4% paraformaldehyde (wt/vol...
N-ChIP on sperm
reagent used in the protocol.
- Use
- N-ChIP was conducted on sperm chromatin using previously described procedures. Briefly, the cauda epidydymis was dissected into 1 ml of M2 medium (Sigma), and sperm were allowed to swim into the medium for 1 h at 37 °C. Five epidydymis were used per sample, and each experimental group had three samples. At lea...
Transgenerational olfactory sensitivity after F0 conditioning
Studies that have examined the effect of parental stress after conception, either in utero or postnatally, have often found an anxiogenic phenotype in the offspring,. Using an elevated plus maze to assay for anxiety-like behavior, we found that prior, rather limited foot shock conditioning of the F0 generation, di...
- Use
- Studies that have examined the effect of parental stress after conception, either in utero or postnatally, have often found an anxiogenic phenotype in the offspring,. Using an elevated plus maze to assay for anxiety-like behavior, we found that prior, rather limited foot shock conditioning of the F0 generation, di...
Inheritance of effects in the F2 and IVF-derived generations
When tested in our behavioral assay, F2-Ace-C57 mice exposed to odors for the first time showed increased OPS to acetophenone compared with F2-Prop-C57 mice, whereas F2-Prop-C57 mice showed an increased OPS to propanol ( ). This persistent behavioral sensitivity to the F0-conditioned odor was accompanied by correspo...
- Use
- When tested in our behavioral assay, F2-Ace-C57 mice exposed to odors for the first time showed increased OPS to acetophenone compared with F2-Prop-C57 mice, whereas F2-Prop-C57 mice showed an increased OPS to propanol ( ). This persistent behavioral sensitivity to the F0-conditioned odor was accompanied by correspo...
Altered epigenetic signature at Olfr151 (M71) locus in sperm
Given that our data suggests a biological inheritance of our behavioral and structural effects via parental gametes, we sought to examine sperm of the F0 generation males for epigenetic clues that might explain an enhanced representation for the M71 receptor ( ). CpG methylation is one mechanism by which a particula...
- Use
- Given that our data suggests a biological inheritance of our behavioral and structural effects via parental gametes, we sought to examine sperm of the F0 generation males for epigenetic clues that might explain an enhanced representation for the M71 receptor ( ). CpG methylation is one mechanism by which a particula...
Altered epigenetic signature at Olfr151 (M71) locus in sperm
Published data also support the idea of epigenetic marking in sperm by indicating that sperm-associated histones are retained with chromatin of the paternal genome at the one-cell embryo stage,. To investigate the possibility that histone modifications mark the Olfr151 (M71) locus, we collected sperm from F0-Ace a...
- Use
- Published data also support the idea of epigenetic marking in sperm by indicating that sperm-associated histones are retained with chromatin of the paternal genome at the one-cell embryo stage,. To investigate the possibility that histone modifications mark the Olfr151 (M71) locus, we collected sperm from F0-Ace a...
Behavior
All behavior was performed in a double-blind manner and data acquired using automated computer software programs. We are grateful to S. Banerjee, R. Andero-Gali, D. Choi, J. Goodman and F. Morrison for help with ensuring double-blindness of data acquisition and analysis.
- Use
- All behavior was performed in a double-blind manner and data acquired using automated computer software programs. We are grateful to S. Banerjee, R. Andero-Gali, D. Choi, J. Goodman and F. Morrison for help with ensuring double-blindness of data acquisition and analysis.
Elevated plus maze
The elevated-plus maze consists of an elevated platform with two walled, closed arms and two non-walled, open arms connected by an open center. The mice were placed onto the center between the plus maze arms and were recorded exploring the plus maze for 5 min. The amount of time spent in the closed and open arms is...
- Use
- The elevated-plus maze consists of an elevated platform with two walled, closed arms and two non-walled, open arms connected by an open center. The mice were placed onto the center between the plus maze arms and were recorded exploring the plus maze for 5 min. The amount of time spent in the closed and open arms is...
Olfactory fear conditioning of parents
Mice were trained to associate acetophenone or propanol presentation with mild foot shocks. For this purpose, the Startle-Response system (SR-LAB, San Diego Instruments) was modified to deliver discrete odor stimuli as previously described. The mice were trained on 3 consecutive days, with each training day consist...
- Use
- Mice were trained to associate acetophenone or propanol presentation with mild foot shocks. For this purpose, the Startle-Response system (SR-LAB, San Diego Instruments) was modified to deliver discrete odor stimuli as previously described. The mice were trained on 3 consecutive days, with each training day consist...
Auditory fear conditioning
Mice were pre-exposed to sound attenuated conditioning chambers (San Diego Instruments) for three consecutive days before training. On the day of auditory fear conditioning, mice received five conditioned-unconditioned stimulus pairings (conditioned stimulus: 30-s, 6-kHz, 75-dB tone; unconditioned stimulus: 500-ms,...
- Use
- Mice were pre-exposed to sound attenuated conditioning chambers (San Diego Instruments) for three consecutive days before training. On the day of auditory fear conditioning, mice received five conditioned-unconditioned stimulus pairings (conditioned stimulus: 30-s, 6-kHz, 75-dB tone; unconditioned stimulus: 500-ms,...
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Transgenerational olfactory sensitivity after F0 conditioning
Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue. In our case, to assay for behavioral sensitivity to an odor, we used a modified FPS protocol that consists of odor presentation before the startle stimuli. An odor-potentiated startle (OPS) score is computed, in which an enhanced OPS reflects a greater startle to the odor relative to control, when the odor is paired with the startle stimulus. Traditionally, FPS tests have been used to query the emotional state of the animal and the valence of the stimulus paired with the startle. It is important to note that we did not use this test as a measure of valence of the odor, but rather as a readout of the sensitivity toward that odor, similar to FPS tests that have been used to test the sensitivity of mice to natural...
Elevated plus maze
The elevated-plus maze consists of an elevated platform with two walled, closed arms and two non-walled, open arms connected by an open center. The mice were placed onto the center between the plus maze arms and were recorded exploring the plus maze for 5 min. The amount of time spent in the closed and open arms is viewed as a measure of anxiety.
OPS of adult offspring
Mice were habituated to the startle chambers for 5-10 min on three separate days. On the day of testing, mice were first exposed to 15 startle-alone (105-dB noise burst) trials (leaders), before being presented with ten odor + startle trials randomly intermingled with ten startle-alone trials. The odor + startle trials consisted of a 10-s odor presentation co-terminating with a 50-ms, 105-dB noise burst. For each mouse, an OPS score was computed by subtracting the startle response in the first odor + startle trial from the startle response in the last startle-alone leader. This OPS score was then divided by the last startle-alone leader and multiplied by 100 to yield the percent OPS score (% OPS) reported in the results. Mice were exposed to the acetophenone-potentiated startle (acetophe-none + startle) and propanol-potentiated startle (propanol + startle) procedures on independ...
Odor sensitivity
Mice were placed in a three-chambered box and allowed to explore between all three chambers for 10 min. A particular concentration of odor (, either acetophenone or propanol) contained in a 1.5-ml Eppendorf tube was placed in one of the chambers with the middle chamber empty and an empty Eppendorf tube in the farthest chamber. Association time with either the odor or the empty chamber was recorded. An aversion index was computed by subtracting the amount of time spent in the open chamber from the time spent in the odor chamber. Pilot experiments on independent mice revealed an increasing aversion for either acetophenone or propanol as the concentration increased. Independent mice were used in the acetophenone and propanol experiments (F1-Ace-C57, n = 16; F1-Prop-C57, n = 16).
IVF
IVF was carried out by the Emory Transgenic Mouse Facility (TMF) located in a different building from our colony ( http://med.emory.edu/research/core_labs/transgenic_mouse/ ) across the Emory Campus. Briefly, F0-M71 males were fear conditioned either to acetophenone or propanol as outlined above. 10 d after fear conditioning, sperm was collected from the caudal epididymis and vas deferens of these males in our facility, and then transported to the TMF wherein IVF was conducted by TMF personnel blinded to the experimental conditions of the sperm samples according to protocols followed by Jackson Laboratory. In vitro fertilization culture medium, Mouse Vitro Fert (MVF, Cook Medical), was used for sperm isolation, IVF and zygote culture. Superovulated C57BL/6 female mice were used as oocyte donors. Sperm were co-incubated with oocytes in MVF for 4 h in a 5% CO 2 incubator, the presumpti...
β-galactosidase staining, quantitation of MOE OSN number and glomerular area in bulb
The MOE and olfactory bulbs of 2-month-old M71-LacZ mice were processed for β-galactosidase staining, and then M71 OSN number and glomerular area were quantitated using previously published protocols. Briefly, lateral whole-mount MOE and brains were rapidly dissected and placed into 4% paraformaldehyde (wt/vol) for 10 min at ~23 °C, after which they were washed three times in 1 × phosphate-buffered saline (PBS) for 5 min. M71-LacZ was stained for β-galactosidase using 45 mg of X-gal (1 mg ml -1 ) dissolved in 600 µl of DMSO and 45 ml of a solution of 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl in 1 × PBS, and incubated at 37 °C for 3 h.
N-ChIP on sperm
N-ChIP was conducted on sperm chromatin using previously described procedures. Briefly, the cauda epidydymis was dissected into 1 ml of M2 medium (Sigma), and sperm were allowed to swim into the medium for 1 h at 37 °C. Five epidydymis were used per sample, and each experimental group had three samples. At least 3 × 10 6 sperm were used for each ChIP. Sperm were then collected by centrifugation at 4 °C for 10 min at 500 g, and resuspended in 1 × PBS, 1 mM PMSF. Sperm were then lysed on ice for 10 min in 1 × PBS, 1 mM PMSF, 0.5% Triton X-100 (vol/vol). Nuclei were pelleted by centrifugation at 4 °C for 10 min at 371 g. The pellet was then suspended in 1 × PBS, 1 mM PMSF, 10 mM DTT, and incubated at 37 °C for 30 min, before the addition of 0.6 mM CaCl 2 and MNase (Sigma) to yield mono-, di- and tri-nucleosomal chromatin. Immunoprecipitation was th...
Measurement outputs
What raw and processed outputs should exist?
Studies that have examined the effect of parental stress after conception, either in utero or postnatally, have often found an anxiogenic phenotype in the offspring,. Using an...
- 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
Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue....
- 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
These data suggest that the effect of paternal olfactory fear conditioning on neuroanatomy is associated with increased numbers of OSNs and increased glomerular area, both speci...
- 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 address this issue, and to address potential maternal transmission, we conducted a cross-fostering study. Sexually naive female mice were conditioned with acetophenon...
- 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
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Previously, we reported that the behavioral response (increased FPS to acetophenone) of F0-Ace conditioned males is complemented by an increase in the number of acetophenone-responsive M71-expressing OSNs in the MOE and M71 glomerular area in the olfactory bulbs.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Studies that have examined the effect of parental stress after conception, either in utero or postnatally, have often found an anxiogenic phenotype in the offspring,. Using an...; Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue....; These data suggest that the effect of paternal olfactory fear conditioning on neuroanatomy is associated with increased numbers of OSNs and increased glomerular area, both speci...; To further address this issue, and to address potential maternal transmission, we conducted a cross-fostering study. Sexually naive female mice were conditioned with acetophenon....
from paperStatistical comparison
Previously, we reported that the behavioral response (increased FPS to acetophenone) of F0-Ace conditioned males is complemented by an increase in the number of acetophenone...
from paperReporting output
Report representative outputs alongside summary comparisons for Studies that have examined the effect of parental stress after conception, either in utero or postnatally, have often found an anxiogenic phenotype in the offspring,. Using an..., Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue...., These data suggest that the effect of paternal olfactory fear conditioning on neuroanatomy is associated with increased numbers of OSNs and increased glomerular area, both speci..., To further address this issue, and to address potential maternal transmission, we conducted a cross-fostering study. Sexually naive female mice were conditioned with acetophenon....
inferred from protocolStructured statistical methods
Previously, we reported that the behavioral response (increased FPS to acetophenone) of F0-Ace conditioned males is complemented by an increase in the number of acetophenone...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue. In our case, to assay for behavioral sensitivity to an odor, we used a modified FPS protocol that consists of odor presentation before the startle stimuli. An odor-potentiated startle (OPS) score is computed, in which an enhanced OPS reflects a greater startle to the odor relative to control, when the odor is paired with the startle stimulus. Traditionally, FPS tests have been used to query the emotional state of the animal and the valence of the stimulus paired with the startle. It is important to note that we did not use this test as a measure of valence of the odor, but rather as a readout of the sensitivity toward that odor, similar to FPS tests that have been used to test the sensitivity of mice to natural odors such as fox urine. Enhanced OPS to acetophenone in our experiment would be interpreted as an enhanced behavioral sensitivity to acetophenone, not necessarily an increase in fear to acetophenone. Making any statements about valence specificity and the emotional value of the odor would necessi...
The elevated-plus maze consists of an elevated platform with two walled, closed arms and two non-walled, open arms connected by an open center. The mice were placed onto the center between the plus maze arms and were recorded exploring the plus maze for 5 min. The amount of time spent in the closed and open arms is viewed as a measure of anxiety.
Mice were habituated to the startle chambers for 5-10 min on three separate days. On the day of testing, mice were first exposed to 15 startle-alone (105-dB noise burst) trials (leaders), before being presented with ten odor + startle trials randomly intermingled with ten startle-alone trials. The odor + startle trials consisted of a 10-s odor presentation co-terminating with a 50-ms, 105-dB noise burst. For each mouse, an OPS score was computed by subtracting the startle response in the first odor + startle trial from the startle response in the last startle-alone leader. This OPS score was then divided by the last startle-alone leader and multiplied by 100 to yield the percent OPS score (% OPS) reported in the results. Mice were exposed to the acetophenone-potentiated startle (acetophe-none + startle) and propanol-potentiated startle (propanol + startle) procedures on independent days in a counter-balanced fashion.
Mice were placed in a three-chambered box and allowed to explore between all three chambers for 10 min. A particular concentration of odor (, either acetophenone or propanol) contained in a 1.5-ml Eppendorf tube was placed in one of the chambers with the middle chamber empty and an empty Eppendorf tube in the farthest chamber. Association time with either the odor or the empty chamber was recorded. An aversion index was computed by subtracting the amount of time spent in the open chamber from the time spent in the odor chamber. Pilot experiments on independent mice revealed an increasing aversion for either acetophenone or propanol as the concentration increased. Independent mice were used in the acetophenone and propanol experiments (F1-Ace-C57, n = 16; F1-Prop-C57, n = 16).
IVF was carried out by the Emory Transgenic Mouse Facility (TMF) located in a different building from our colony ( http://med.emory.edu/research/core_labs/transgenic_mouse/ ) across the Emory Campus. Briefly, F0-M71 males were fear conditioned either to acetophenone or propanol as outlined above. 10 d after fear conditioning, sperm was collected from the caudal epididymis and vas deferens of these males in our facility, and then transported to the TMF wherein IVF was conducted by TMF personnel blinded to the experimental conditions of the sperm samples according to protocols followed by Jackson Laboratory. In vitro fertilization culture medium, Mouse Vitro Fert (MVF, Cook Medical), was used for sperm isolation, IVF and zygote culture. Superovulated C57BL/6 female mice were used as oocyte donors. Sperm were co-incubated with oocytes in MVF for 4 h in a 5% CO 2 incubator, the presumptive zygotes were washed, and were cultured overnight in a 150-µl MVF drop in the incubator. In the second morning, two-cell embryos were scored and washed in MVF drop; pseudopregnant CD-1 female mice of 9-13 weeks of age were used as embryo recipients. 15-20 embryos were transferred...
The MOE and olfactory bulbs of 2-month-old M71-LacZ mice were processed for β-galactosidase staining, and then M71 OSN number and glomerular area were quantitated using previously published protocols. Briefly, lateral whole-mount MOE and brains were rapidly dissected and placed into 4% paraformaldehyde (wt/vol) for 10 min at ~23 °C, after which they were washed three times in 1 × phosphate-buffered saline (PBS) for 5 min. M71-LacZ was stained for β-galactosidase using 45 mg of X-gal (1 mg ml -1 ) dissolved in 600 µl of DMSO and 45 ml of a solution of 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl in 1 × PBS, and incubated at 37 °C for 3 h.
N-ChIP was conducted on sperm chromatin using previously described procedures. Briefly, the cauda epidydymis was dissected into 1 ml of M2 medium (Sigma), and sperm were allowed to swim into the medium for 1 h at 37 °C. Five epidydymis were used per sample, and each experimental group had three samples. At least 3 × 10 6 sperm were used for each ChIP. Sperm were then collected by centrifugation at 4 °C for 10 min at 500 g, and resuspended in 1 × PBS, 1 mM PMSF. Sperm were then lysed on ice for 10 min in 1 × PBS, 1 mM PMSF, 0.5% Triton X-100 (vol/vol). Nuclei were pelleted by centrifugation at 4 °C for 10 min at 371 g. The pellet was then suspended in 1 × PBS, 1 mM PMSF, 10 mM DTT, and incubated at 37 °C for 30 min, before the addition of 0.6 mM CaCl 2 and MNase (Sigma) to yield mono-, di- and tri-nucleosomal chromatin. Immunoprecipitation was then carried out as described for the MOE and followed the previously established protocol. The antibodies were used at 1:1,000 and were specific to H3 trimethyl lysine-27 (07-449) and acetyl histone H3 (06-599) from Upstate. Immunoprecipitated DNA was isolated by phenol-chloroform extraction and eth...
Machine-readable layer
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"name": "Parental olfactory experience influences behavior and neural structure in subsequent generations methods",
"description": "Evidence-backed execution summary for Parental olfactory experience influences behavior and neural structure in subsequent generations methods from Parental olfactory experience influences behavior and neural structure in subsequent generations.",
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"name": "Transgenerational olfactory sensitivity after F0 conditioning",
"text": "Fear-potentiated startle (FPS) is a behavioral test to assay for fear learning. FPS manifests as an augmented startle response in the presence of the aversive conditioned cue. In our case, to assay for behavioral sensitivity to an odor, we used a modified FPS protocol that consists of odor presentation before the startle stimuli. An odor-potentiated startle (OPS) score is computed, in which an enhanced OPS reflects a greater startle to the odor relative to control, when the odor is paired with the startle stimulus. Traditionally, FPS tests have been used to query the emotional state of the animal and the valence of the stimulus paired with the startle. It is important to note that we did not use this test as a measure of valence of the odor, but rather as a readout of the sensitivity toward that odor, similar to FPS tests that have been used to test the sensitivity of mice to natural..."
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"name": "Elevated plus maze",
"text": "The elevated-plus maze consists of an elevated platform with two walled, closed arms and two non-walled, open arms connected by an open center. The mice were placed onto the center between the plus maze arms and were recorded exploring the plus maze for 5 min. The amount of time spent in the closed and open arms is viewed as a measure of anxiety."
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"name": "OPS of adult offspring",
"text": "Mice were habituated to the startle chambers for 5-10 min on three separate days. On the day of testing, mice were first exposed to 15 startle-alone (105-dB noise burst) trials (leaders), before being presented with ten odor + startle trials randomly intermingled with ten startle-alone trials. The odor + startle trials consisted of a 10-s odor presentation co-terminating with a 50-ms, 105-dB noise burst. For each mouse, an OPS score was computed by subtracting the startle response in the first odor + startle trial from the startle response in the last startle-alone leader. This OPS score was then divided by the last startle-alone leader and multiplied by 100 to yield the percent OPS score (% OPS) reported in the results. Mice were exposed to the acetophenone-potentiated startle (acetophe-none + startle) and propanol-potentiated startle (propanol + startle) procedures on independ..."
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"text": "Mice were placed in a three-chambered box and allowed to explore between all three chambers for 10 min. A particular concentration of odor (, either acetophenone or propanol) contained in a 1.5-ml Eppendorf tube was placed in one of the chambers with the middle chamber empty and an empty Eppendorf tube in the farthest chamber. Association time with either the odor or the empty chamber was recorded. An aversion index was computed by subtracting the amount of time spent in the open chamber from the time spent in the odor chamber. Pilot experiments on independent mice revealed an increasing aversion for either acetophenone or propanol as the concentration increased. Independent mice were used in the acetophenone and propanol experiments (F1-Ace-C57, n = 16; F1-Prop-C57, n = 16)."
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"text": "IVF was carried out by the Emory Transgenic Mouse Facility (TMF) located in a different building from our colony ( http://med.emory.edu/research/core_labs/transgenic_mouse/ ) across the Emory Campus. Briefly, F0-M71 males were fear conditioned either to acetophenone or propanol as outlined above. 10 d after fear conditioning, sperm was collected from the caudal epididymis and vas deferens of these males in our facility, and then transported to the TMF wherein IVF was conducted by TMF personnel blinded to the experimental conditions of the sperm samples according to protocols followed by Jackson Laboratory. In vitro fertilization culture medium, Mouse Vitro Fert (MVF, Cook Medical), was used for sperm isolation, IVF and zygote culture. Superovulated C57BL/6 female mice were used as oocyte donors. Sperm were co-incubated with oocytes in MVF for 4 h in a 5% CO 2 incubator, the presumpti..."
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"name": "β-galactosidase staining, quantitation of MOE OSN number and glomerular area in bulb",
"text": "The MOE and olfactory bulbs of 2-month-old M71-LacZ mice were processed for β-galactosidase staining, and then M71 OSN number and glomerular area were quantitated using previously published protocols. Briefly, lateral whole-mount MOE and brains were rapidly dissected and placed into 4% paraformaldehyde (wt/vol) for 10 min at ~23 °C, after which they were washed three times in 1 × phosphate-buffered saline (PBS) for 5 min. M71-LacZ was stained for β-galactosidase using 45 mg of X-gal (1 mg ml -1 ) dissolved in 600 µl of DMSO and 45 ml of a solution of 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl in 1 × PBS, and incubated at 37 °C for 3 h."
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"name": "N-ChIP on sperm",
"text": "N-ChIP was conducted on sperm chromatin using previously described procedures. Briefly, the cauda epidydymis was dissected into 1 ml of M2 medium (Sigma), and sperm were allowed to swim into the medium for 1 h at 37 °C. Five epidydymis were used per sample, and each experimental group had three samples. At least 3 × 10 6 sperm were used for each ChIP. Sperm were then collected by centrifugation at 4 °C for 10 min at 500 g, and resuspended in 1 × PBS, 1 mM PMSF. Sperm were then lysed on ice for 10 min in 1 × PBS, 1 mM PMSF, 0.5% Triton X-100 (vol/vol). Nuclei were pelleted by centrifugation at 4 °C for 10 min at 371 g. The pellet was then suspended in 1 × PBS, 1 mM PMSF, 10 mM DTT, and incubated at 37 °C for 30 min, before the addition of 0.6 mM CaCl 2 and MNase (Sigma) to yield mono-, di- and tri-nucleosomal chromatin. Immunoprecipitation was th..."
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