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Results
Parturition, growth and development
Repeated challenge with aerosolized OVA produced a robust inflammatory response, observed in maternal sera and bronchoalveolar lavage fluid (BALF), with the characteristic allergy/asthma cytokine profile previously described in mice39, 40, 41, 42 (see Supplementary Materials). To determine whether this OVA-induced immune activation during pregnancy resulted in altered parturition rates, a chi-square analysis was performed between MAA and PBS-exposed dams for the number of full-term, aborted and cannibalized litters. Out of 27 initial litters, only two litters were cannibalized following parturition (one PBS and one MAA) and two additional pregnancies did not come to term (two PBS, zero MAA). The majority of pregnancies (11 PBS and 12 MAA) were full term with no significant differences between treatment groups, χ2(2,n=27)=2.074, P=0.355.
Offspring born to mothers with repeated exposure to allergic asthma treatment (that is, MAA group) showed altered weight and length developmental trajectories throughout juvenile development (Figure 1). Offspring of both treatment groups showed significant increases in body length as a main effect of developmental day, F(6,74)=325.434, P0.001; however, offspring of MAA-exposed dams were significantly longer throughout all developmental time points, F(1,213)=79.054, P0.001. Moreover, repeated measures analysis of variance revealed a significant day × treatment interaction for pup length, F(6,74)=2.286, P=0.044 (Figure 1a). Similarly, although offspring of both MAA and PBS-exposed dams exhibited significant weight gain throughout the initial three weeks of life, F(6,64)=216.012, P=0.001, offspring of MAA-exposed dams weighed significantly more than PBS controls as indicated by a main effect for treatment, F(1,198)=45.992, P=0.0001 (Figure 1b). This weight gain was independent of developmental day, F(6,64)=1.165, P=0.336. Interestingly, by 10 weeks of age, these differences in body mass were no longer present between treatment groups, F(1,27)=0.110, P=0.742 (Figure 1c).
Figure 1.
Developmental growth trajectories of offspring born to PBS and MAA-exposed dams. (a) Pups born to mothers exposed to MAA were significantly longer throughout juvenile development compared with typically developing control mice. (b) Similarly, offspring of MAA dams weighed significantly more than control pups throughout development. (c) Differences in weight between treatment groups were no longer present by adulthood at 10 weeks of age. *P0.05. MAA, maternal allergic asthma; PBS, phosphate-buffered saline.
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Social behaviors
During the initial habituation phase of the social approach task, there was no baseline preference for either empty chamber between treatment groups: main effect chamber, F(1,162)=0.002, P=0.969; main effect treatment, F(1,162)=0.036, P=0.850; chamber by treatment interaction, F(1,162)=0.381, P=0.538, indicating no inherent bias at the start of the behavioral task. During the subsequent social approach phase, there was a significant main effect for chamber preference, with mice spending significantly more time in the mouse chamber compared with the object chamber F(1,162)=65.979, P=0.0001 (Figure 2a). This preference for the social chamber was apparent in both treatment groups as indicated by a null effect for treatment, F(1,162)=0.272, P=0.603 and treatment by chamber interaction F(1,162)=0.606, P=0.438. Specifically, both MAA and PBS offspring spent significantly more time in the chamber with the mouse compared with the chamber with the novel object. However, analysis of the strength of the social preference between treatment groups revealed a significant decrease in sociability in MAA offspring compared with PBS control offspring F(1,79)=4.225, P=0.043. Offspring of MAA-exposed dams had a 33% decrease in the sociability score, indicating a deficit in social behavior compared with offspring of PBS-exposed dams (Figure 2b). This difference in sociability was independent of sex, main effect: F(1,79)=0.421, P=0.518; sex by treatment interaction: F(1,79)=0.836, P=0.363. Deficits in sociability between MAA and PBS groups were brought on by a significant increase in the time spent with the novel object by offspring of MAA dams, F(1,79)=4.079, P=0.047, as well as a trend towards a decrease in the time spent with the novel mouse, F(1,79)=3.590, P=0.062 (Figure 2c).
Figure 2.
MAA results in social behavior deficits in offspring. (a) Offspring of both MAA and control dams showed significant chamber preference for the novel mouse, although the magnitude of preference was reduced in MAA offspring. (b) Mice born to mothers repeatedly exposed to allergy/asthma displayed significantly reduced sociability scores compared with control mice from PBS-treated dams. (c) Social behavior deficits in MAA offspring were a result of reductions in the total time spent exploring the chamber containing the novel mouse and increases in total time spent exploring the chamber with the novel object. (d) On average, mice from both treatment groups won half of all tube test challenges, indicating no differences in social dominance between groups. *P0.05, #P=0.06. MAA, maternal allergic asthma; PBS, phosphate-buffered saline.
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No significant differences were observed in social dominance between PBS and MAA groups (Figure 2d). Analysis of variance revealed no difference in the percentage of matches won between treatment groups F(1,26)=0.709, P=0.407. In fact, offspring from both MAA-exposed dams and control PBS-exposed dams won ~50% of all tube test bouts. The average number of wins was independent of sex as revealed by a null effect for sex, F(1,26)=0.061, P=0.806, as well as no sex by treatment interaction, F(1,26)=0.061, P=0.806.
Repetitive and perseverative behaviors
In mice, marble-burying is used as an index of perseverative digging behavior analogous to the restricted, repetitive patterns of behavior observed in neurodevelopmental disorders.43 Offspring of MAA-exposed dams buried significantly more marbles compared with offspring of PBS control dams (Figure 3a), as indicated by a main effect for treatment F(1,78)=4.854, P=0.031. Interestingly, a significant main effect for sex was observed, F(1,78)=6.340, P=0.014, with male mice burying a greater percentage of marbles compared with females. This sex difference was independent of treatment as noted by a null sex by treatment interaction: F(1,78)=2.074, P=0.154.
Figure 3.
Alterations in stereotypical restricted/repetitive behaviors. (a) Offspring of MAA dams buried significantly more marbles than control mice. (b) There was a significant reduction in the total time spent grooming observed in mice born to mothers repeatedly challenged with OVA throughout pregnancy. MAA, maternal allergic asthma; PBS, phosphate-buffered saline.
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The total time spent grooming during a 10-min period was significantly reduced in offspring of MAA-exposed dams compared with typically developing mice born to control dams (Figure 3b). When controlling for variations across litters, linear mixed effects model revealed a significant main effect of treatment for grooming times F(1,81)=4.21, P=0.043. On average, there was a 25% reduction in grooming time for offspring of MAA-exposed dams (mean=44.36, s.d.=34.30) compared with offspring of PBS-exposed dams (mean=60.41, s.d.=36.49). Interestingly, there was a significant main effect for sex, F(1,52)=5.380, P=0.024, with males spending significantly more time grooming compared with females. This sex-specific difference was apparent regardless of treatment group as observed by a null sex by treatment interaction, F(1,52)=0.038, P=0.846.
Hypo/hyperactivity
To determine whether phenotypic differences between offspring of MAA- and PBS-exposed dams were due to hyper/hypoactivity, mice were placed in an open arena and measured for locomotor behavior. Between treatment groups, no differences were observed in either the total distance traveled, F(1,27)=0.001, P=0.994 (Figure 4a), or average velocity, F(1,27)=0.165, P=0.688, demonstrating similar locomotor activity between offspring of MAA- and PBS-exposed dams (Figure 4b). Moreover, there were no main effects for sex (distance traveled, F(1,27)=0.538, P=0.496; average velocity, F(1,27)=0.317, P=0.578) and no sex by treatment interactions (distance traveled, F(1,27)=0.037, P=0.849; average velocity, F(1,27)=0.004, P=0.849).
Figure 4.
Measures of locomotor activity, memory formation and anxiety. No differences were observed in (a) the total distance traveled or (b) average velocity between treatment groups during the open-field observations. (c) Mice from both MAA- and PBS-exposed dams showed normal object recognition as measured by a preference (that is, 50% sniffing) for a novel versus familiar object. (d) There were no differences observed in the percent of time spent in the open arms of the elevated plus maze arena between treatment groups. (e) Similarly, no differences were observed in the total number of entries into the open arm between offspring of PBS-treated and MAA dams. (f) These similarities parallel similarities in the total number of entries into all arms of the maze, an internal control for locomotor activity. MAA, maternal allergic asthma; PBS, phosphate-buffered saline.
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Learning and memory
To determine whether reductions in social behavior are due to deficits in cognition, offspring from MAA- and PBS-exposed dams were measured for alterations in memory performance using the novel-object recognition task. One day following the memory acquisition phase, offspring of MAA- and PBS-exposed dams spent significantly more time exploring the novel object, as indicated by a novel-object exploration time 50% (one-sample t-test): PBS, t(15)=3.569, P=0.003; MAA, t(15)=3.967, P=0.001. There were no significant differences in the percentage of time spent exploring the novel object between sex, F(1,27)=1.608, P=0.216 or treatment groups, F(1,27)=0.003, P=0.954, suggesting equal memory formation between offspring of PBS and MAA dams (Figure 4c).
Anxiety-like behavior
There was no significant difference in the percentage of time spent in the open arms of the elevated plus maze between treatment groups, F(1,49)=1.789, P=0.187, as well as no difference between sex F(1,49)=0.018, P=0.892 or sex by treatment interactions F(1,49)=0.182, P=0.671, indicating an absence of a high anxiety-like phenotype in offspring of MAA-exposed dams (Figure 4d). Moreover, offspring of both MAA and PBS-exposed dams made equal number of entries into the open arms of the maze F(1,49)=0.796, P=0.377 (Figure 4e) as well as similar number of total entries into any arm F(1,49)=0.589, P=0.447 (Figure 4f).
Serotonin transporter expression
The analysis of serotonin transporter protein levels in the cortex of MAA and PBS offspring revealed a significant main effect for treatment, F(1,12)=9.67, P=0.011. Mice born to dams exposed to MAA had a 33% increase in the expression of SERT compared with control offspring of PBS-exposed dams (Figure 5). These differences did not differ between males and females as indicated by a null effect for sex, F(1,12)=0.678, P=0.429, as well as no sex by treatment interactions, F(1,12)=0.066, P=0.803. Increases in SERT expression in the cortex positively correlated with marble-burying behavior, rs(14)=0.602, P=0.023, with mice expressing the highest SERT levels burying the greatest number of marbles. These correlations between SERT expression and behavior were not evident in any other behavior measured.
Figure 5.
Immunoblot analysis of serotonin transporter (SERT) protein expression in the cortex. Mice born from mothers sensitized to OVA before pregnancy and repeatedly challenged with aerosolized OVA during pregnancy had significantly greater expression of SERT in the cortex compared with typically developing mice born to PBS control dams. *P0.05. MAA, maternal allergic asthma; OVA, ovalbumin; PBS, phosphate-buffered saline.
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