The Role of Redox Dysregulation in the Effects of Prenatal Stress on Embryonic Interneuron Migration

Abstract

Maternal stress during pregnancy is associated with increased risk of psychiatric disorders in offspring, but embryonic brain mechanisms disrupted by prenatal stress are not fully understood. Our lab has shown that prenatal stress delays inhibitory neural progenitor migration. Here, we investigated redox dysregulation as a mechanism for embryonic cortical interneuron migration delay, utilizing direct manipulation of pro- and antioxidants and a mouse model of maternal repetitive restraint stress starting on embryonic day 12. Time-lapse, live-imaging of migrating GAD67GFP+ interneurons showed that normal tangential migration of inhibitory progenitor cells was disrupted by the pro-oxidant, hydrogen peroxide. Interneuron migration was also delayed by in utero intracerebroventricular rotenone. Prenatal stress altered glutathione levels and induced changes in activity of antioxidant enzymes and expression of redox-related genes in the embryonic forebrain. Assessment of dihydroethidium (DHE) fluorescence after prenatal stress in ganglionic eminence (GE), the source of migrating interneurons, showed increased levels of DHE oxidation. Maternal antioxidants (N-acetylcysteine and astaxanthin) normalized DHE oxidation levels in GE and ameliorated the migration delay caused by prenatal stress. Through convergent redox manipula-tions, delayed interneuron migration after prenatal stress was found to critically involve redox dysregulation. Redox biology during prenatal periods may be a target for protecting brain development.

Keywords: N-acetylcysteine; antioxidants; interneuron migration; oxidative stress; prenatal stress.

Figure 1.

Time-lapse imaging of migrating interneurons in acutely excised forebrain tissue slices. (A–C) Arrow indicates direction of migrating GABAergic progenitor cells and arrowheads indicate the starting position and end position of each interneuron after 30 min. (A) Tissue section of E14 embryo in artificial cerebral spinal fluid (aCSF) at beginning time point (t = 0 min). (B) Tissue section pictured in A at t = 30 min. (C) Magnification of A and B with mash-up of time points 0 and 30 min. Schematic of migration of 10 interneuron progenitors after approximately 1–1.5 h of E14 embryo in aCSF (D–G) and aCSF with either 1 μM H₂O₂ (H–J) or 5 μM H₂O₂ (K). Arrow indicates direction of migration stream and dot is end-point. (L) Bar graph shows increased average velocity of GAD67GFP+ cells and (M) depicts increased average degree of deviation from migration stream with H₂O₂ exposure. (N) Schematic of hypothesized migration delay showing average velocity and average angle deviation for each of the 5 live imaging sessions. For the purpose of calculating migration deviation, we designated interneurons confined to migration stream as deviating 0°. Dashed lines represent the boundaries of the tissue slices. (**P < 0.01 by 2-tailed paired t-test, n = 5 embryos, aCSF and H₂O₂ groups).

Figure 2.

Rotenone increased levels of DHE oxidation and delayed migration in the E14 brain. ICV-injected rotenone increased levels of DHE oxidation in E14 ganglionic eminence (GE) tissue compared with ICV-injected saline, as measured by dihydroethidium (DHE) mean fluorescent intensity (MFI) of cells and normalized to MFI = 1.00 (A, B, G). Prenatal stress (PS) delayed progenitor migration at E14 (D) compared with nonstressed (C) embryonic neocortical tissue (H). ICV-injected rotenone (F) also delayed migration of GAD67GFP+ cells at E14 compared with ICV-injected saline (E, H). For the DHE experiments, ICV Saline: n = 5, and ICV Rotenone: n = 5 and Mann–Whitney U test for hypothesis testing (*P < 0.05). For the migration experiments, NS: n = 10, PS: n = 4, ICV Saline: n = 8, and ICV Rotenone: n = 8. (*P < 0.05 compared with ICV saline by paired t-test, ^$P < 0.05 compared with NS by paired t-test).

Figure 3.

Prenatal stress (PS) altered the expression of some genes involved in redox regulation. Only Tr1 (A) among antioxidant activity genes (A–G) was altered of other genes related to redox regulation (H–K), only Nrf2 and Aifm1 were altered. (NS = nonstressed; NS: n = 20, PS: n = 20; *P < 0.05, **P < 0.01 by unpaired t-tests correcting for multiple comparisons using Bonferroni method).

Figure 4.

Prenatal stress (PS) disrupted glutathione stores and dysregulated antioxidant enzyme activity. (A) Ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG) showed a trend increase as the result of prenatal stress (vs. nonstress [NS], P = 0.05 by a priori t-test), but showed a main effect of NAC treatment (^##P < 0.01 by 2-way ANOVA) and an interaction of stress and treatment (^ϕP < 0.05). (B) A main effect of NAC (^#P < 0.05) on GSH. (C) A significant baseline difference was observed between nonstress and prenatal stress control brains (**P < 0.01), and main effects of NAC (^##P < 0.01) and stress (^αP < 0.05), as well as an interaction of stress and NAC (^ϕP < 0.05) was found in GSSG brains. (D) A trend decrease in GPx1 activity resulting from prenatal stress (vs. NS, P = 0.06 by a priori t-test) and a main effect of NAC (^##P < 0.01) was detected. (E) A trend decrease in TR1 activity was found between NS and PS embryonic forebrains (P = 0.06 by a priori t-test). For glutathione experiments, NS: n = 6, PS: n = 6, NS NAC: n = 10, and PS NAC: n = 7. For GPx1 experiment, NS: n = 12, PS: n = 14, NS NAC: n = 3, and PS NAC: n = 3. For TR1 experiment, NS: n = 12, and PS: n = 14.

Figure 5.

Prenatal stress (PS) increased levels of DHE oxidation in the ganglionic eminence (GE) of the embryonic brain. (A) All analyses were normalized to mean fluorescent intensity, MFI = 1.00 of control (Con) tissue. Measurements of MFI were taken with ×40 objective images. (B) PS increased MFI (*P < 0.05 compared with NS). In the absence of PS, MFI in NS NAC (C) and NS AST (E) were reduced below the levels of PS control (**P < 0.01). PS NAC (D) and PS AST (F) decreased levels of MFI (**P < 0.01 compared with PS control). (G) Antimycin-A (AA) was used as the positive control and showed the highest DHE oxidation (^$$P < 0.01 by a priori t-test compared with NS control). (Kruskal–Wallis and Dunn’s tests run for NAC and AST exposure effects separately).

Figure 6.

Delays in GABAergic progenitor cell migration after PS normalized by maternal antioxidants. (A) NS control migration, (B) NS NAC migration, and (C) NS AST migration. (D) PS control migration was significantly delayed compared with NS control (^$P < 0.05 by a priori t-test) and NS saline (micrograph not shown; quantitative data shown in G). (E) PS NAC migration was significantly increased compared with PS control (**P < 0.01) and a main effect of NAC (^##P < 0.01) was revealed. (F) PS AST migration was significantly increased compared with PS control (**P < 0.01) and a main effect of AST (^##P < 0.01) was found. A main effect of stress (^αP < 0.05) was also unveiled between PS embryos (D–F) and NS embryos compared with (A–C). The interaction of stress and NAC treatment (^ϕϕP < 0.01) was also significant (G). (H) Quantitative PCR of the Cxcr4 gene, coding for a receptor expressed by migrating inhibitory progenitor cells (P = 0.06) (2-way ANOVAs run for NAC and AST exposure effects separately). For migration: NS: n = 10, Saline: n = 10, PS: n = 8, NS NAC: n = 10, PS NAC: n = 10, NS AST: n = 8, and PS AST: n = 9. For Cxcr4 gene expression: NS: n = 17 and PS: n = 19.