Elsevier

Neuroscience

Volume 199, 29 December 2011, Pages 535-547
Neuroscience

Regeneration, Repair, and Developmental Neuroscience
Research Paper
Dexamethasone induces apoptosis in the developing rat amygdala in an age-, region-, and sex-specific manner

https://doi.org/10.1016/j.neuroscience.2011.09.052Get rights and content

Abstract

Exposure to glucocorticoids (GCs) in early development can lead to long-term changes in brain function and behavior, although little is known about the underlying neural mechanisms. Perinatal exposure to GCs alters adult anxiety and neuroendocrine responses to stress. Therefore, we investigated the effects of either late gestational or neonatal exposure to the GC receptor agonist dexamethasone (DEX), on apoptosis within the amygdala, a region critical for emotional regulation. DEX was administered to timed-pregnant rat dams from gestational day 18 until parturition, or postnatal day 4–6. Offspring were sacrificed the day following the last DEX treatment, and tissue was processed for immunohistochemical detection of cleaved caspase-3, a marker for apoptotic cells. Prenatal DEX treatment significantly increased the number of cleaved caspase-3–positive cells in the amygdala of both sexes, largely due to increases within the medial and basomedial subregions. Postnatal DEX treatment also increased cleaved caspase-3 immunoreactivity within the amygdala, although effects reached significance only in the central nucleus of females. Overall, DEX induction of cleaved caspase-3 in the amygdala was greater following prenatal compared with postnatal treatment, yet in both instances, elevations in cleaved caspase-3 correlated with an increase in pro-apoptotic Bax mRNA expression. Dual-label immunohistochemistry of cleaved caspase-3 and the neuronal marker NeuN confirmed that virtually all cleaved caspase-3–positive cells in the amygdala were neurons, and a subset of these cells (primarily following postnatal treatment) expressed a GABAergic calcium-binding protein phenotype (calbindin or calretinin). Together these results indicate that early developmental GC exposure induces neuronal apoptosis within the amygdala in an age-, sex-, and region-dependent manner.

Highlights

▶Dexamethasone induces apoptosis in the perinatal amygdala. ▶Effects are timing, sex, and subregion dependent. ▶Dependent on increases in Bax expression.

Section snippets

Animals

Timed-pregnant Sprague–Dawley dams were obtained from Charles River Laboratories (Wilmington, MA, USA) at 7 days' gestation and were handled for 4 days (GD14–17) before treatment. Dams were singly housed in a vivarium with a 14/10-h light/dark cycle, lights on at 0600 h, with food and water available ad libitum. For prenatal treatment, dams were s.c. injected with DEX dissolved in ethanol (2%) and diluted in safflower oil (0.4 mg/kg) or ethanol/safflower oil alone once daily at 1000 h on

Brain and body weights

Separate two-way ANOVAs revealed that both pre- and postnatal DEX treatments decreased brain [prenatal tx: F(1,20)=34.35, P<0.001; postnatal tx: F(1,20)=21.19, P<0.001] and body weights [prenatal tx: F(1,20)=65.57, P<0.001; postnatal tx: F(1,20)=13.9, P<0.01]. No significant sex or interaction effects were found for either treatment period. Post hoc comparisons revealed that body weights taken at the time of euthanasia were significantly decreased in both males and females following both DEX

Discussion

In this series of studies, we examined the effects of both late gestational and postnatal exposure to DEX on neural cell death. The results of these studies show that brain and body weight were decreased in male and female offspring of DEX-treated dams. Postnatal treatment of rats also caused overall decreases in brain and body weight. Of importance, DEX treatment had significant effects on the degree of cleaved caspase-3–positive cells in the amygdala, and these effects occurred in an age-,

Acknowledgments

The authors acknowledge Drs. Stuart Tobet and Jill Goldstein for providing helpful input in the design of these studies and the preparation of the manuscript. Also thanks to Alicia Quihuis and Anthony Lacaganina for their expert technical assistance. Support for these studies was provided by United States Public Health Service grants NS039951 and MH082679 (R.J.H.).

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      These control and experimental rat groups received daily i.p. injections of either DEX (0.4 mg/kg dissolved in 0.1 ml pyrogen-free saline) or an equi-volume of pyrogen-free saline starting from GD14 until GD21 respectively. The DEX dose was used because prenatal exposure to this dose was shown to affect brain plasticity in cortical areas of adult rats (Hiroi et al., 2016; Zuloaga et al., 2011, 2012). All injections were performed in the morning (09:00 and 11:00).

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