Alterations in behavior in adult offspring mice following maternal inflammation during pregnancy

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  • Alterations in Behavior in AdultOffspring Mice FollowingMaternal InflammationDuring Pregnancy

    Golan Hava

    Lev Vered

    Mazar YaelDepartment of Developmental Molecular

    Genetics and ZlotowskiCenter for Neuroscience

    Ben-Gurion University of the NegevBeer-Sheva, Israel


    Hallak MordechaiDepartment of Obstetrics and Gynecology

    Faculty of Health Sciences, Ben-GurionUniversity of the Negev, Beer-Sheva, Israel

    Huleihel MahoudDepartment of Microbiology and

    Immunology and Cancer Research CenterBen-Gurion University of the Negev

    Beer-Sheva, Israel

    ABSTRACT: Maternal intrauterine inflammation during pregnancy poses a majorthreat of neurodevelopmental brain damage in offspring and may cause poorcognitive and perceptual outcomes. In mice, we have previously shown thatmaternal inflammation induced by lipopolysaccharide (LPS) at gestation day 17thincreased the levels of the pro-inflammatory cytokine IL-6 in the fetal brain. In thisstudy, we used the same system and examined the effect of short, systemic maternalinflammation on anxiety and social behavior of the offspring. Adult offspring fromthe maternal inflammation group showed increased anxiety, as indicated by theelevated plus maze. Social interaction among offspring from the test groups wasexamined when two unfamiliar mice from different litters were introduced into anew home-cage. Offspring from the maternal inflammation group showed reducedactivity, indicating increased fear. In addition, offspring from the maternalinflammation group were less aggressive towards their cagemates and they spenta significantly longer time trimming the whiskers of their cagemates during the first30 min of their interaction, compared to offspring from the control group. Our datasuggest that short systemic maternal inflammation have long-lasting consequenceson the adult mouse stress and social behavior. 2006 Wiley Periodicals, Inc.Dev Psychobiol 48: 162168, 2006.

    Keywords: cytokines; neurogenesis; social interactions; anxiety; prenatal stress;mouse


    Intrauterine inflammation during pregnancy increases the

    risk of preterm delivery, neurodevelopmental brain

    damage, neurological disorders, and mental retardation

    in the offspring (Dammann, Kuban, & Leviton, 2002;

    Saliba & Henrot, 2001). Intrauterine infection may affect

    the immature brain by the induction of proinflammatory


    The proinflammatory cytokines, tumor necrosis factor-

    alpha (TNFa), interleukin-1 (IL-1) and IL-6 were

    associated with intrauterine infection, preterm delivery,

    neonatal infections, and neonatal brain damage. In the

    brain, these proteins are expressed in both glial cells and

    neurons. In addition to their function in the immune

    response, these cytokines modulate neuron development

    and function. TNFa is involved in the regulation of neuritegrowth (Golan, Levav, Mendelsohn, & Huleihel, 2004;

    Neumann et al., 2002), affects neuronal survival (Barker,

    Middleton, Davey, & Davies, 2001; Yang, Lindholm,

    Konishi, Li, & Shen, 2001), and regulates AMPA receptor

    expression (Beattie et al., 2002). IL-1b and TNFaare involved in the regulation of synaptic plasticity

    (Butler, OConnor, & Moynagh, 2004; Cunningham,

    Murray, ONeill, Lynch, & OConnor, 1996; Schneider

    et al., 1998; Tancredi et al., 1992). In the animal model,

    administration of lipopolysaccharide (LPS) to pregnant

    rats increased the expression of TNFa and IL-1b mRNAin a fetal brain in a dose-dependent manner. In addition,

    Received 20 May 2005; Accepted 26 August 2005Correspondence to: Golan HavaPublished online in Wiley InterScience

    ( DOI 10.1002/dev.20116

    2006 Wiley Periodicals, Inc.

  • decreased myelin basic protein expression and enhanced

    expression of the astrocytes marker; glial fibrillary acidic

    protein, was observed a week after application; (Cai,

    Pan, Pang, Evans, & Rhodes, 2000). LPS administration

    to pregnant rats upregulated the mRNA expression of the

    stress-related peptide, corticotrophin-releasing hormone

    (CRH) in the fetal brain (Gayle et al., 2004), suggesting

    the possibility of inducing a fetal stress response.

    Activation of the stress response during pregnancy was

    shown to have long-term consequences on the response of

    adult offspring to stressful situations, as demonstrated in

    rodents, primates, and humans (Breivik et al., 2002;

    Clarke & Schneider, 1993; Kofman, 2002; Saliba &

    Henrot, 2001).

    In our previous study, we demonstrated that a single

    intraperitoneal (i.p) administration of LPS (0.12 mg/g) to

    pregnant mice on gestation day 17 (E17), produced

    significantly increased IL-6 levels in the maternal spleen

    and the fetal brain, (3 hr after the injection), compared to

    the controls treated with saline (Golan, Lev, Hallak,

    Sorokin, & Huleihel, 2005). Here, we examine the long-

    term effects of this treatment on stress-related behavior in

    the adult offspring.


    Study Design

    Jackson Black C-57 mice were used. Pregnant mice were

    treated on gestation day 17 (17 days after the day of

    mating). Mice were randomly assigned to one of two

    groups: (1) saline injections (i.p)control group (n 12),(2) E. Coli LPS injection (.12 mg/g mouse/100 mL, i.p)test group (n 12). This LPS dose was shown to inducematernal inflammation without inducing preterm deliv-

    ery. Measurements of IL-6 in maternal spleen indicate

    maternal inflammatory response, which last up to 6 hr

    after the LPS injection (data not shown).

    The mouse colony was kept on a 12:12 hr light/dark

    schedule; food and water were provided ad libitum. At the

    age of 3 weeks, offspring were separated from their

    mothers and housed in isolation, in separate home cages:

    20 cm wide, 35 cm long, 15 cm high, until the age of

    8 months, when they were given behavioral tests. Two

    offspring from each mother, male and female, were

    included in the elevated plus maze experiment and a

    single male offspring from each litter was included in the

    social-interaction study. All the procedures were per-

    formed according to the Principles of laboratory care

    (NIH publication no. 8623, revised 1985) as well as the

    guidelines from the Israeli Council on Animal Care and

    were approved by the Ben-Gurion University of the Negev

    Animal Care and Use Committee.

    Behavior Examination

    After a week of daily handling (5 min a day) by the

    researcher blind to experimental groups, the following

    behavioral examinations were performed:

    Elevated Plus Maze. A plus maze with 40 cm long, 10 cmwidth, and 15 cm high arms was elevated 50 cm above

    the floor and used to measure the time (in seconds) spent

    in the open versus the closed arms and the number of

    crossing between the closed and open arms during 5 min

    of examination (Lister, 1987).

    Social Interaction. Social interactions between unfa-miliar offspring from the same treatment group were

    examined in four pairs (each pair in a separate cage). The

    mice were housed in separate cages after weaning and

    until they were observed. Three episodes of 30 min each

    were recorded and analyzed at three time-periods

    (relative to the time when the mice were introduced)

    0, 24 hr, and 7 days, all during the dark cycle.

    The observations began the moment that two offspring

    were introduced simultaneously into a new cage (day 1,

    time 05). Mice behavior was videotaped for later

    analysis. Each episode was analyzed for various home

    cage behaviors (Lijam et al., 1997). The duration (in

    seconds) of playing, resting huddled, whisker trimming,

    fighting, and the number of times that a mouse sniffed the

    anogenitals of its cagemate, or attacked its cagemate

    from the rear, or dug tunnels. In addition, we also

    measured a general parameter: general activitythe

    number of times mice crossed a line along the midline of

    the cage, this measure is affected by both: locomotion

    and the interactions between the cagemates. In four cases

    a mouse was severely wounded by its cagemate between

    the second and third observation periods. In these cases,

    the adversarial mice were separated and were not studied

    during the last time period (the 7th day following their

    first interaction).

    Statistical Analysis

    We used SPSS software for our statistical analyses. The

    results of the elevated plus maze were analyzed by means

    of the 2-way ANOVA for the effect of treatment and sex.

    Changes in the behavior of the offspring during the social

    interactions observed were evaluated for changes, each

    parameter both within the group and between the groups

    (2-way ANOVA for repeated measurements).


    The elevated plus maze was used to evaluate the possible

    effect of maternal inflammation during pregnancy on

    Prenatal Maternal Inflammation Reduced Anxiety 163

  • anxiety-related behavior in the offspring. Offspring

    behavior in the elevated plus maze showed that maternal

    inflammation during pregnancy significantly increased

    the preference of the offspring to stay in the close

    arms, indicated by the ratio of the time spent in the close

    arms versus the time spent in the open arms. This ratio was

    2.09 .14 for offspring from the maternal inflammationgroup, compared to 1.63 .18 in offspring from thecontrol group (n 24 and 21, respectively, p .03,F 4.6 between groups, no significant effect of sex wasdetected p .38, F .76, Fig. 1A). No significantdifferences were observed in the number of entries into

    the close arms. Animals from the control group entered

    30 2.4 times, while the test group entered 33.17 1.7times during the 5 min of the test, as demonstrated in

    Figure 1B.

    Figure 2A demonstrates that during the 30 min of

    observation on the first day, offspring of the control

    group did not play with each other at all. Although not

    significant (p .68, F .17) offspring of the maternalinflammation group did playing for a short time during

    the initial day. The duration of positive interactions, such

    as playing, was increased on the second day of paired

    housing and during the last observation of offspring

    from the maternal inflammation group. Huddling

    was observed only at the end of the last session in

    both groups, no significant differences were observed

    (p .86, F .03, Fig. 2B). In contrast, the anogenitalsniffing frequency was higher in the beginning and

    declined over time. In both groups, a significant

    reduction in the number of anogenital sniffing interac-

    tions was observed within the first day (p< .03, controland p< .004, LPS). Overall, the number of anogenitalsniffing events showed a similar trend in offspring from

    both groups (p< .17, F 2.04). An equal tendency was

    FIGURE 1 Effect of maternal inflammation mice anxiety in elevated plus maze. (A) The ratio

    between the time mice spent in the close arms versus the time mice spent in the open arms is

    presented. (B) The number of entries into the close arms. n(control) 21, and n(maternalinflammation ) 24, p .03, F 4.6 between groups, no significant effect of sex was detectedp .38, F .76, p .03,F 4.6 between groups, no significant effect of sex was detected p .38,F .76, possibly due to small number of female subjects.

    FIGURE 2 Effect of maternal inflammation on social interac-

    tions. (A) Playing, p .68, F .17; (B) huddling, p .86,F .03; (C) nose to back, p .17, F 2.04. (Group X episode:control, p .29; LPS, p< .001). Analyses of the differentparameters are presented for 5-min observations: during the first

    030 min of paired housing on the first day and during the second

    and last observation periods at 24 hr and 7 days later.

    164 Hava et al.

  • observed when the general activity of the mice was

    compared, as depicted in Figure 3A. A similar significant

    reduction over time in general activity was observed in

    both groups, (Group X episode: control, p .059; LPS,p .01; and between groups p .1), compared to the offspring fromthe maternal inflammation group that adapted during the

    week (p< .02). In general, the frequency of interactionsand the amount of activity or neutral interaction was

    reduced over time during the week in both groups, and

    the only significant difference found between the groups

    was in the time spent trimming whiskers.

    Aggressive behavior, such as chasing, frequently

    evolved following anogenital sniffing. In such cases,

    chasing developed into an attack from the rear, followed

    by fighting. Chasing was observed on the first day, but

    diminished significantly during the first day in offspring

    of both groups as depicted in Figure 4A (Group X

    episode: control, p .008; LPS, p< .017). No significantdifference was observed between the groups for this

    parameter (p> .1, F .2). A high frequency of attacksfrom the rear was observed on the first day in both groups.

    The number of attacks was significantly reduced in

    offspring of both the control and the maternal inflamma-

    tion groups (Group X episode: control, p .004; LPS,p< .003). Like the time spent chasing, the frequency ofsuch observed attacks was equal in both groups (Fig. 4B.

    p .8, F .05). The time that offspring spent fightingwas, in general, higher on the first day. However, no clear

    tendency was observed over time. Offspring from the

    control group spent more time fighting, as compared to

    offspring from the maternal inflammation group. How-

    ever, differences in this parameter did not reach

    significance (Fig. 4C. p< .1, F 2.7). Although aggres-sive behavior diminished significantly during the first

    day of observation, in three out of five cages in the control

    group, one mouse was severely wounded between the

    2nd and the 7th day of paired housing. In contrast, only

    one mouse (out of four pairs) was wounded in the

    maternal inflammation group.


    Our main findings in the present study demonstrate that 8-

    month-old mice, that had experienced an inflammatory

    response during the last stages of their embryonic

    development, E17, had a distinct response to stressful

    situations, mainly confirmed by the longer time spent in

    FIGURE 3 Effect of maternal inflammation on social

    interactions. (A) Activity, p .5, F .3 (Group X episode:control, p .059; LPS, p .01); (B) digging, p .6,F .21; (C)whisker trimming, p .0001, F 28.5 (Group X episode:control, p .68; LPS, p .023). Analyses of the differentparameters are presented for 5-min observations during the first

    030 min of paired housing on the first day and for the second

    and last observation periods at 24 hr and 7 days later.

    Prenatal Maternal Inflammation Reduced Anxiety 165

  • the close arms of the elevated plus maze and a tendency of

    reduced aggressiveness towards their cagemates.

    Hyperanxiety in the elevated plus maze was observed

    in adult rodents previously exposed to prenatal stress (PS).

    This response was induced by different type of stressors,

    such as immobilization (Takahashi, Turner, & Kalin,

    1992), cold-water immersion (Velazquez-Moctezuma,

    Dominguez Salazar, & Cruz Rueda, 1993), noise and

    flashing lights (Fride & Weinstock, 1988), and additional

    paradigms (Weinstock, 1997; Weinstock, Fride, &

    Hertzberg, 1988). It is well documented that the stress

    response was associated with modifications in the

    hypothalamus-pituitary-adrenal (HPA) axis. Alterations

    of the maternal HPA axi...


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