Parental olfactory experience influences behavior and neural structure in subsequent generations methods
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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...
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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...
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