Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb 13;10(2):e0117680.
doi: 10.1371/journal.pone.0117680. eCollection 2015.

Prenatal stress down-regulates Reelin expression by methylation of its promoter and induces adult behavioral impairments in rats

Ismael Palacios-García  1 , Ariel Lara-Vásquez  2 , Juan F Montiel  3 , Gabriela F Díaz-Véliz  4 , Hugo Sepúlveda  5 , Elías Utreras  6 , Martín Montecino  5 , Christian González-Billault  6 , Francisco Aboitiz  2
Affiliations

Prenatal stress down-regulates Reelin expression by methylation of its promoter and induces adult behavioral impairments in rats

Ismael Palacios-García et al. PLoS One. .

Abstract

Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new insights on the molecular mechanisms behind the effects of prenatal stress.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Experimental design and reference map.
A, Experimental protocol of prenatal restraint stress. B, Map of neuronal quantification. The frontal (A), parietal (B), and caudal (C) cortical levels were subdivided in a medial (red square; 1), dorsal (blue square; 2) and dorsolateral (green square; 3) tiers. E = embryonic days; P = postnatal days.
Fig 2
Fig 2. Validation of stress in the pregnant rat.
A, The average adrenal gland weights were higher in stressed dams with respect to controls. B, analogously, in stressed rats the average body weight gain was 22.8% less than in controls. Values are mean ± SEM, n = 7 rats per group.
Fig 3
Fig 3. Cerebral cortex reelin and NeuN immunohistochemistry.
Microphotographies of the dorsal tier of frontal (A, C, E, G), and retrosplenial (B, D, F, H) cortical levels. Control brains (A, B) show numerous clusters of Cajal-Retzius neurons while the stress groups (C, D) show only few isolated Cajal-Retzius neurons. Total neuronal density is show in Control brains (E, F) and stress group (G, H). No differences between groups are observed. Scale bars = 50 μm.
Fig 4
Fig 4. Prenatal stress induces global decrease of reelin expressing neurons density in the prenatal (E20) rat brain (Left side) and no changes of NeuN neurons density (Right side).
Immunohistochemistry results: All graphs show the immunoreactive neurons expressed in neurons/mm3. Opens bars represent control group and filled bars represent stress group. A, B and C, show the results of frontal, parietal and retrosplenial cortex respectively. Values are mean ± SEM, n = 7–10 rats per group (reelin analysis) and n = 3 rats per group (NeuN analysis).
Fig 5
Fig 5. In vivo (A) and in vitro (B) expression of Reelin and downstream elements in control and stress conditions analyzed by Western Blot.
The left side of the panel shows the result of density pixel quantification, the right side shows a representative example of each protein expression. Expression levels observed in vivo are maintained after 5 DIV. Values are mean ± SEM. n = 8–12 per group at in vivo experiments, n = 3 per group at in vitro experiment. (c) Cdk5 kinase activity measured from protein extract from brain cortex of control (n = 5) and stressed (n = 4) rats. All data are presented as the mean and SEM. * p < 0.05.
Fig 6
Fig 6. In vitro (A) and in vivo (C) analyses of DNA methylation at the reelin gene promoter region in samples from control and stress groups.
Genomic DNA obtained from both groups was cleaved with DNA methylation sensitive restriction enzyme HpaII and then amplified by conventional PCR using specific primers against a reelin promoter region containing an HpaII site (-786/-625). Undigested genomic DNA is included as amplification control in all PCR reactions. Efficient digestion of DNA samples using the HpaII enzyme was controlled by analyzing a region devoid of DNA methylation at the Ric-8B gene promoter containing several HpaII cleavage sites (digestion control). A region of the Runx2 gene promoter lacking HpaII sites was selected to show equal sample loading in each lane of the gels. Digestion with the isoschizomer restriction enzyme MspI (DNA methylation insensitive) was also performed to control for efficient cleavage at the reelin promoter sequence analyzed (Data not shown). DNA methylation differences between control and stress rats groups measured in vivo (B) and in vitro (D) were quantified by determining changes in pixel density at the bands amplified by PCR and visualized through conventional DNA electrophoresis (see methods). (E), diagram depicting the selected upstream promoter region with its putative methylation sites. The reelin promoter has several transcription site starts over the CpG island. Upstream the promoter is the HpaII/MspI restriction site that was selected for our analyses. Values are expressed as relative ratios between PCR amplifications of the reelin promoter sequence after digestion with HpaII over the amplifications of the Runx2 control promoter sequence (loading control). Values represent a mean ± SEM. N = 3 per group. *p<0.05.
Fig 7
Fig 7. Prenatal stress increases the spontaneous rearing (A) and spontaneous locomotor activity (B) responses in young adult rats (P60).
Values are mean ± SEM, n = 11 rats per group. C and D, Prenatal stress decreases the total time in the open arms and does not affect the total number of entries of open and closed arms in the elevated plus-maze. Values are mean ± SEM, n = 11 rats per group.
Fig 8
Fig 8
A, Passive avoidance learning retention test in rats. There were significant differences in latency time between control and prenatally stressed rats, on days four and five post shock. B, Morris maze test in rats. There were no significant differences in latency time between control and prenatally stressed rats. Each point represents mean ± SEM, n = 9 rats per group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dayan J, Creveuil C, Dreyfus M, Herlicoviez M, Baleyte JM, et al. (2010) Developmental model of depression applied to prenatal depression: role of present and past life events, past emotional disorders and pregnancy stress. PLoS One 5: e12942 10.1371/journal.pone.0012942 - DOI - PMC - PubMed
    1. Andrus BM, Blizinsky K, Vedell PT, Dennis K, Shukla PK, et al. (2010) Gene expression patterns in the hippocampus and amygdala of endogenous depression and chronic stress models. Mol Psychiatry. - PMC - PubMed
    1. Calabrese F, Molteni R, Racagni G, Riva MA (2009) Neuronal plasticity: a link between stress and mood disorders. Psychoneuroendocrinology 34 Suppl 1: S208–216. 10.1016/j.psyneuen.2009.05.014 - DOI - PubMed
    1. Weinstock M (2001) Alterations induced by gestational stress in brain morphology and behaviour of the offspring. Prog Neurobiol 65: 427–451. - PubMed
    1. Seckl JR, Holmes MC (2007) Mechanisms of disease: glucocorticoids, their placental metabolism and fetal’programming’ of adult pathophysiology. Nat Clin Pract Endocrinol Metab 3: 479–488. - PubMed

Publication types

MeSH terms