Dexamethasone induces apoptosis in the developing rat amygdala in an age-, region-, and sex-specific manner

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.

Figure 1. Cleaved caspase-3 immunoreactive cells in the amygdala following prenatal and postnatal dexamethasone treatment

Pregnant dams were treated with DEX or oil from gestation day 18-21 (prenatal tx) and pups euthanized on the day of birth (P0), or neonates were treated from postnatal day 4-6 (postnatal tx) and euthanized on postnatal day 7 (P7). The number of cleaved caspase-3 immunoreactive cells was counted in the amygdala and are expressed as number of cells per tissue section examined. At least 3 sections were examined per individual. Each bar represents the mean +/- SEM. * Post hoc comparisons indicate p<.05 compared to same sex Oil vehicle treatment. N=5-6 per group.

Figure 2. Cleaved caspase-3 immunoreactive cells within amygdala sub-regions following prenatal dexamethasone treatment

The number of cleaved caspase-3 immunoreactive cells was counted in the medial (A), central (B), basomedial (C), or basolateral (D) amygdala following prenatal treatment with DEX. Representative images of cleaved caspase-3 immunoreactive cells are shown in the medial and basomedial amygdala in P0 rats following treatment with DEX (E,G) or oil vehicle (F,H) on GD18-22. Images were captured using a 20× objective in cresyl violet counterstained sections. * Post hoc comparisons indicate p<.05 compared to same sex Oil treatment. N=5 per group. Scale bar= 200μm.

Figure 3. Cleaved caspase-3 immunoreactive cells within amygdala sub-regions following postnatal dexamethasone treatment

The number of caspase-3 immunoreactive cells was quantitated in the medial (A), central (B), basomedial (C), and basolateral (D) amygdala regions at P7 following postnatal treatment (P4-6) with DEX. Each bar represents the mean +/- SEM. * Post hoc comparisons indicate p<.05 compared to same sex Oil vehicle treatment. N=5-6 per group.

Figure 4. Bax and Bcl2 mRNA in the amygdala following pre- and postnatal dexamethasone treatment

Bax (A,B) and Bcl2 (C,D) mRNA expression in microdissected amygdala excised from prenatal (P0) and postnatal (P7) DEX and vehicle treated rats. Bax mRNA expression was significantly increased in DEX compared to vehicle treatment groups; * p<.05. N=3-7 per group for prenatal treatment and N=5-8 per group for postnatal treatment. MOIL, male oil vehicle treated; FOIL, female oil vehicle treated; MDEX, male DEX treated; FDEX, female DEX treated.

Figure 5. Co-localization of cleaved caspase-3 with markers for neurons and calcium binding proteins in dexamethasone and vehicle treated rats

(A) The percentage of cleaved caspase-3 immunoreactive cells co-expressing the neuronal marker NeuN. (B) The percentage of cleaved caspase-3 immunoreactive cells co-expressing calbindin and calretinin at P7 (postnatal tx). * Post hoc comparisons indicate p<.05 compared to same sex Oil treatment. N=3 per group for NeuN/caspase-3. N=5 per group for calbindin/caspase-3 and calretinin/caspase-3. MOIL, male oil vehicle treated; FOIL, female oil vehicle treated; MDEX, male DEX treated; FDEX, female DEX treated.

Figure 6. Photomicrographs showing immunofluorescent co-localization of caspase-3 and NeuN, calbindin, and calretinin

(A,D,G,J) Photomicrographs showing representative cells which express cleaved caspase-3 (red). (B) NeuN, (E) calbindin, and (H) calretinin positive cells (green). DAPI nuclear label is shown in blue (K). Merged photographs which reveal co-localization of caspase-3 with NeuN, calbindin, calretinin, and DAPI (C,F,I,L). Note the relatively weak NeuN labeling of the caspase-3 positive cell (B) indicating a decreased NeuN phenotype in this apoptotic neuron. Images are captured using a 40× objective. White arrow indicates co-localized cells; yellow arrow indicates a caspase-3 positive cell that is not co-localized; red arrow indicates a fragmented nucleus with condensed chromatin.