Article Figures & Data
Figures
- Figure 1.
Timeline for the behavioral and metabolic studies. PND = postnatal day.
- Figure 2.
Social behavior results at PND21. A, Frequency of interacting with stranger 1 in T2. B, Time spent interacting with stranger 1 in T2. C, Frequency of grooming in T2. D, Time spent grooming in T2. E, Frequency of interacting with stranger 1 in T3. F, Time spent interacting with stranger 1 in T3. G, Frequency of interacting with stranger 2 in T3. H, Time spent interacting with stranger 2 in T3. I, Frequency of grooming in T3. J, Time spent grooming in T3. The F test was used for data analysis. Treatment, sex, and treatment by sex interaction were included in the model. Treatment effects were analyzed with dam nested under treatment and used as error term. There was significant (p < 0.05) interaction between treatment and sex in frequency and time spent in grooming in T3. Significant differences are indicated in the figures. Error bars represent SEM. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group. Datapoints for all replicates who engaged in the various behaviors are shown. For grooming in T2, many of the animals in both groups did not exhibit this behavior.
- Figure 3.
Audible and ultrasonic communications at PND21. A, Fraction of calls in the ultrasonic range (above 20 kHz). B, Average power of calls below 20 kHz. C, Average power of calls above 20 kHz. D, Power of calls. The F test was used for data analysis. Treatment, sex, and treatment by sex interaction were included in the model. Treatment effects were analyzed with dam nested under treatment and used as error term. There was significant (p < 0.01) treatment effect on fraction of calls above 20 kHz as indicated in A. Error bars represent SEM. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group. Datapoints for all replicates who engaged in the various behaviors are shown. However, many of the animals in both groups did not exhibit any calls during the trial period.
- Figure 4.
Social behavior results at adulthood. A, Frequency of interacting with stranger 1 in T2. B, Time spent interacting with stranger 1 in T2. C, Frequency of grooming in T2. D, Time spent grooming in T2. E, Frequency of interacting with stranger 1 in T3. F, Time spent interacting with stranger 1 in T3. G, Frequency of interacting with stranger 2 in T3. H, Time spent interacting with stranger 2 in T3. I, Frequency of grooming in T3. J, Time spent grooming in T3. The F test was used for data analysis. Treatment, sex, and treatment by sex interaction were included in the model. Treatment effects were analyzed with dam nested under treatment and used as the error term. There were significant (p ≤ 0.05) treatment effects on frequency and time spent in interacting with Stranger 1 in T3 as indicated in E, F. Error bars represent SEM. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group.
- Figure 5.
Audible and ultrasonic communications at adulthood. A, Fraction of calls in the ultrasonic range (above 20 kHz). B, Average power of calls below 20 kHz. C, Average power of calls above 20 kHz. D, Power of calls. The F test was used for data analysis. Treatment, sex, and treatment by sex interaction were included in the model. Treatment effects were analyzed with dam nested under treatment and used as error term. There was significant (p = 0.05) treatment effect on average power of calls as indicated in D. Error bars represent SEM. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group. As with Figures 2 and 3, datapoints for all animals who engaged in these behaviors are shown. More animals called at this age than at PND21 to allow for inferences to be made, but some animals remained silent during the trial.
- Figure 6.
Barnes maze results. A, Distance traveled. B, Travel speed. C, Number of head entries into escape hole. D, Time spent around escape hole. E, Likelihood of mice locating the correct escape hole. Distanced traveled, velocity, number of head entries into escape hole, and time spent around escape hole were analyzed as a split plot in space and time. Statistical model contained the fixed effects of treatment, sex, day, and all possible interactions with treatment, sex, and day. Latency was analyzed by using the PROC PHREG and proportional hazard ratio functions of SAS. A result <1 indicates that the treatment group is less likely to locate the correct escape hole compared with the CTL group. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group.
- Figure 7.
EPM results. A, Number of times spent immobile. B, Duration of time spent immobile. C, Number of entries into open arms. D, Time spent in open arms. E, Number of entries into closed arms. F, Time spent in closed arms. The F test was used for data analysis. Treatment, sex, and treatment by sex interaction were included in the model. Treatment effects were analyzed with dam nested under treatment and used as error term. There was significant (p = 0.01) treatment effect on number of times (A) and duration of time (B) spent immobile. Error bars represent SEM. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group.
- Figure 8.
Indirect calorimetry and body mass results. A, Total distance traveled during the dark cycle. B, Percentage of time remaining still during the dark cycle. C, Food intake during the dark cycle. D, Body mass. E, Total distance traveled during the light cycle. F, Percentage of time remaining still during the light cycle. G, Food intake during the light cycle. Data were analyzed with nested design and dam as experimental unit for treatment effects with treatment, sex, light cycle, and interactions between each of these factors as main effects. To illustrate possible sex differences, data are plotted separately for each sex. Significant differences are indicated in the figures. Number of replicates tested = 13 female and 13 male mice for CTL group, 10 female and 10 male mice for OXY group.
- Figure 9.
Gene expression in hippocampal tissue. Gene expression data, as determined by qPCR assay, were normalized by using combined average dCt values of the two housekeeping genes: B2m and Rpl7 and then analyzed based on treatment, sex, and their interactions, and dam was considered the experimental unit for treatment effects. Significant differences are indicated in the figures. Gene expression patterns for: A) Htr2a; B) Oprd1; C) Oprk1; D) Oprl1; E) Oprm1; F) Slc6a4. Number of replicates tested = seven female and seven male mice for CTL group, five female and five male mice for OXY group. Additional qPCR hippocampal results are included in Extended Data Figure 9-1.
- Figure 10.
Gene expression in hypothalamic tissue. Gene expression data, as determined by qPCR assay, were normalized by using combined average dCt values of the two housekeeping genes: B2m and Rpl7 and then analyzed based on treatment, sex, and their interactions, and dam was the considered experimental unit for treatment effects. Significant differences are indicated in the figures. Gene expression patterns for: A) Avp; B) Dnmt3a; C) Oprd1; D) Oprk1; E) Oprl1; F) Oprm1. Number of replicates tested = seven female and seven male mice for CTL group, five female and five male mice for OXY group. Additional qPCR hippocampal results are included in Extended Data Figure 10-1.
- Figure 11.
Circos plot correlations for OXY-exposed versus CTL female mice. mixOmics R program (v6.1.1) was used to correlate Barnes maze, social, EPM, indirect calorimetry, echoMRI, and gene expression data. Correlation cutoff was set to 0.75. Red lines in the center indicate a positive correlation (A), whereas blue lines (B) indicate a negative correlation. Lines on the outside of the circle indicate whether the value was greater in CTL (blue) versus OXY (orange).
- Figure 12.
Circos plot correlations for OXY-exposed versus CTL groups of male mice. mixOmics R program (v6.1.1) was used to correlate Barnes maze, social, EPM, indirect calorimetry, echoMRI, and qPCR testing. Correlation cutoff was set to 0.75. Red lines in the center indicate a positive correlation (A), whereas blue lines (B) indicate a negative correlation. Lines on the outside of the circle indicate whether the value was greater in CTL (blue) versus OXY (orange).
Tables
- Table 1
Primers used for hippocampus and hypothalamus qPCR in male and female CF1 mice offspring
Gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′) Efficiency Avp ATCTGCTGCAGCGACGAGAG TGTACCAGCCTTAGCAGCAG 90.3 B2m1 CCTTCAGCAAGGACTGGTCT TTGATCACATGTCTCGATCCCAG 100.3 Bdnf CGGAGAGCAGAGTCCATTCAG CTCACCTGGTGGAACTTCTTTG 90.7 Cnrip1 AACACTGCAGGTCGAGAACATT TTGCCACACTGTCTCGAAGG 91.3 DAT CTGGAAGATCTGCCCTGTCC CAGCACACCACGCTCAAAAT 103.9 Dnmt3a GATGAGCCTGAGTATGAGGATGG CAAGACACAATTCGGCCTGG 103.6 Dnmt3b CTGTCCGAACCCGACATAGC CCGGAAACTCCACAGGGTA 96.4 Drd1 TTGAGTCCAGGGGTTTTGGG GGGCCTCTTCCTGGTCAATC 99.0 Drd2 TGAACAGGCGGAGAATGGATG GTTTTGCCATTGGGCATGGT 90.0 Esr1 CTTGAACCAGCAGGGTGGC AGGCTTTGGTGTGAAGGGTC 100.2 Esr2 ACGAAGAGTGCTGTCCCAAG GCCAAGGGGTACATACTGGAG 112.0 Gnrh CAACTGTGCTCACCAGCGG TGAGGATCATGTCCACTCTGTTT 110.0 Htr2a CACCGACATGCCTCTCCATT GGCCACCGGTACCCATACAG 90.9 Htr7 GGGACCTGAGGACCACCTAT CAGTGGTCACAGTTTTGTAGCA 98.0 Kiss1 AGACTGTAGACCTGCCCCTT GACTGCTGGCCTGTGGAT 106.7 Lepr CAGAATGACGCAGGGCTGTA GCTCAAATGTTTCAGGCTTTTGGA 92.7 Oprd1 CCGTGGCCTCCGTTTTCC CAATTTGGTGTACCGGACGA 95.0 Oprk1 AGTGCCACCTTCTCGCTTT GGTCTTCATCTTCGTGTATCGGA 99.3 Oprl1 TCTTCGGAGCAGGAGCTAGG GCCACTCAGTACAGTTCCTCC 105.4 Oprm1 CATGGCCCTCTATTCTATCGTGT CCACGTTCCCATCAGGTAGTT 99.4 Oxtr GTCTGGTCAAATACTTGCAGGT CGCGCAGCGAGAAAATGTG 97.6 Rpl7 AGCTGGCCTTTGTCATCAGAA GACGAAGGAGCTGCAGAACCT 96.1 Slc6a4 CTCCGCAGTTCCCAGTACAAG CACGGCATAGCCAATGACAGA 108.5
Extended Data Figure 9-1
Additional gene expression in hippocampal tissue not included in Figure 9. Gene expression data, as determined by qPCR assay, were normalized by using combined average dCt values of the two housekeeping genes: B2m and Rpl7 and then analyzed based on treatment, sex, and their interactions, and dam was considered the experimental unit for treatment effects. Number of replicates tested = seven female and seven male mice for CTL group, five female and five male mice for OXY group. Download Figure 9-1, TIF file.
Extended Data Figure 10-1
Additional gene expression in hypothalamic tissue not included in Figure 10. Gene expression data, as determined by qPCR assay, were normalized by using combined average dCt values of the two housekeeping genes: B2m and Rpl7 and then analyzed based on treatment, sex, and their interactions, and dam was the considered experimental unit for treatment effects. Number of replicates tested = seven female and seven male mice for CTL group, five female and five male mice for OXY group. Download Figure 10-1, TIF file.
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