Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affec

Gestational Chronodisruption Impairs Hippocampal Expression of NMDA Receptor Subunits Grin1b/Grin3a and Spatial Memory in the Adult Offspring

<div><p>Epidemiological and experimental evidence correlates adverse intrauterine conditions with the onset of disease later in life. For a fetus to achieve a successful transition to extrauterine life, a myriad of temporally integrated humoral/biophysical signals must be accurately provided by the mother. We and others have shown the existence of daily rhythms in the fetus, with peripheral clocks being entrained by maternal cues, such as transplacental melatonin signaling. Among developing tissues, the fetal hippocampus is a key structure for learning and memory processing that may be anticipated as a sensitive target of gestational chronodisruption. Here, we used pregnant rats exposed to constant light treated with or without melatonin as a model of gestational chronodisruption, to investigate effects on the putative fetal hippocampus clock, as well as on adult offspring’s rhythms, endocrine and spatial memory outcomes. The hippocampus of fetuses gestated under light:dark photoperiod (12:12 LD) displayed daily oscillatory expression of the clock genes <i>Bmal1</i> and <i>Per2</i>, clock-controlled genes <i>Mtnr1b</i>, <i>Slc2a4</i>, <i>Nr3c1</i> and <i>NMDA</i> receptor subunits 1B-3A-3B. In contrast, in the hippocampus of fetuses gestated under constant light (LL), these oscillations were suppressed. In the adult LL offspring (reared in LD during postpartum), we observed complete lack of day/night differences in plasma melatonin and decreased day/night differences in plasma corticosterone. In the adult LL offspring, overall hippocampal day/night difference of gene expression was decreased, which was accompanied by a significant deficit of spatial memory. Notably, maternal melatonin replacement to dams subjected to gestational chronodisruption prevented the effects observed in both, LL fetuses and adult LL offspring. Collectively, the present data point to adverse effects of gestational chronodisruption on long-term cognitive function; raising challenging questions about the consequences of shift work during pregnancy. The present study also supports that developmental plasticity in response to photoperiodic cues may be modulated by maternal melatonin.</p></div

Effect of gestational chronodisruption and maternal melatonin replacement on daily oscillatory transcription of <i>Per2</i> (a), <i>Bmal1</i> (b), <i>Mtnr1b</i> (c), <i>Slc2a4</i> (d), <i>Nr3c1</i> (e) and NMDA receptors (<i>Grin</i>) subunits (f-h) in the fetal rat hippocampus at day 18 of gestation, from pregnant dams under LL (<i>Red symbols</i>) and LL + Mel (<i>Blue symbols</i>).

<p>Mean ± SEM of 2^−ΔΔCt *: Different from other time points (P<0.05; ANOVA). Note that LL was different to LL + Mel (P<0.05; Two way ANOVA). Grey bars indicate the clock time of melatonin replacement or placebo. In each graph the value at 0400 h is repeated in the next 24 h.</p

Long-term (postnatal day 90) effects of maternal exposure to constant light and maternal melatonin replacement on plasma melatonin (a) and corticosterone (b), spatial memory (c) and on day/night differences in expression of clock genes (d-e), clock controlled genes (f-h) and NMDA receptor subunits (i-k).

<p>Mean ± SEM for plasma hormones, latency time and of 2^−ΔΔCt. 90 days old offspring gestated under LD (<i>Green symbols</i>), LL (<i>Red symbols</i>) and LL + Mel (<i>Blue symbols</i>). #: Different to LD and LL+Mel (P<0.05; Two way ANOVA). *: Different to 11-h (P<0.05; Two way ANOVA).</p

RT-qPCR primers for genes studied<b>.</b>

<p>E: Efficiency assay using fetal and adult hippocampus pooled RNA. a: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091313#pone.0091313-TorresFarfan2" target="_blank">[24]</a>; b: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091313#pone.0091313-Young1" target="_blank">[59]</a>; c:<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091313#pone.0091313-Floyd1" target="_blank">[60]</a>.</p

of melatonin supplementation to pregnant dams kept under constant light on the circadian rhythm of clock and clock-controlled genes in the fetal hippocampus (Mean ± SEM).

<p>N.D.: Not detectable. *: Different to LD (student t test).</p

Effects of gestational condition (LD, LL and LL+Mel) on body weight (g) at birth, 30 and 90 days of age (Mean ± SEM).

<p>Effects of gestational condition (LD, LL and LL+Mel) on body weight (g) at birth, 30 and 90 days of age (Mean ± SEM).</p

Daily oscillatory expression of <i>Per2</i> and <i>Bmal1</i> (a); <i>Mtnr1a</i> and <i>Mtnr1b</i> (b); <i>Nr3c1</i> and <i>Slc2a4</i> (c); NMDA receptor subunit 1B [<i>Grin1b</i>] (d); and 3A-3B [<i>Grin3a</i> and <i>Grin3b</i>] (e), in the fetal rat hippocampus at 18 days of gestational age from pregnant dams maintained in 12:12 LD photoperiod.

<p>Mean ± SEM of 2^−ΔΔCt. *: Different from other time points (P<0.05; ANOVA). Black bars indicate lights-off hours. In each graph the value at 0400 h is repeated in the next 24 h.</p

Effect of maternal exposure to constant light and melatonin replacement on daily rhythm of maternal melatonin (A) and corticosterone (B) at day 18 of gestation.

<p>Mean ± SEM of corticosterone and melatonin measured in maternal plasma collected every 4 hours from 4 pregnant dams maintained under LD (green symbols), 5 LL dams (<i>Red symbols</i>) and 5 LL+Mel dams (<i>Blue symbols</i>). *: Different from other time points for LD (P<0.05; ANOVA and Newman-Keuls). <b>^&</b>: Different from other time points for LL+Mel (P<0.05; ANOVA and Newman-Keuls). <b>^#</b>: Different from other time points for LL (P<0.05; ANOVA and Newman-Keuls). The grey bars indicate the clock time of lights off and melatonin replacement or placebo during subjective night.</p