Binge-like postnatal alcohol exposure triggers cortical gliogenesis in adolescent rats

Abstract

The long-term effects of binge-like postnatal alcohol exposure on cell proliferation and differentiation in the adolescent rat neocortex were examined. Unlike the hippocampal dentate gyrus, where proliferation of progenitors results primarily in addition of granule cells in adulthood, the vast majority of newly generated cells in the intact mature rodent neocortex appear to be glial cells. The current study examined cytogenesis in the motor cortex of adolescent and adult rats that were exposed to 5.25 g/kg/day of alcohol on postnatal days (PD) 4-9 in a binge manner. Cytogenesis was examined at PD50 (through bromodeoxyuridine [BrdU] labeling) and survival of these newly generated cells was evaluated at PD80. At PD50, significantly more BrdU-positive cells were present in the motor cortex of alcohol-exposed rats than controls. Confocal analysis revealed that the majority (>60%) of these labeled cells also expressed NG2 chondroitin sulfate proteoglycan (NG2 glia). Additionally, survival of these newly generated cortical cells was affected by neonatal alcohol exposure, based on the greater reduction in the number of BrdU-labeled cells from PD50 to PD80 in the alcohol-exposed animals compared to controls. These findings demonstrate that neonatal alcohol exposure triggers an increase in gliogenesis in the adult motor cortex.

Figure 1

Schematic of motor cortex region analyzed and experimental design. A) Representative atlas plates from Bregma 0.48 ± 0.3 through −2.12 ± 0.3 used in analyzing the motor cortex. Drawing adapted from Paxinos and Watson (2004). B) Schematic of experimental design described in the Methods section.

Figure 2

Confocal images of BrdU labeling in the adult rat brain. Animals were sacrificed 2 hours after the last BrdU injection. A) NeuN (magenta) labeling B) BrdU (green) labeling and C) the merged image of the labeling in the motor cortex. D) NeuN (magenta) labeling E) BrdU (green) labeling and F) the merged image of the labeling in the hippocampal dentate gyrus. G) NeuN (magenta) labeling H) BrdU (green) labeling and I) the merged image of the labeling in the lateral ventricle. Numerous BrdU-labeled cells were observed throughout the dentate gyrus and lateral ventricle. BrdU-labeled cells were sparse in the motor cortex as compared to the dentate gyrus and lateral ventricle. The effect of a binge-like postnatal alcohol exposure on the cell proliferation and adult neurogenesis in the dentate gyrus was demonstrated in a recent publication by our laboratory (Klintsova et al., 2007). Scale bars = 50 µm in A, B, C, G, H, I; 100 µm in D, E, F. BrdU, bromodeoxyuridine; NeuN, neuronal nuclei.

Figure 3

Postnatal alcohol exposure increased the number of BrdU+ cells on PD 50 in the motor cortex in the AE group compared to SC and SI groups. No difference was found between the SC and SI groups. PD, postnatal day; SC, suckle control; SI, sham intubated; AE, alcohol-exposed. Data expressed as mean ± SEM. *p<0.05 relative to both control groups.

Figure 4

Analyses of BrdU phenotype in the motor cortex. Sections of MC stained with BrdU (green) and A) NeuN (magenta), B) Iba1 (magenta), C) GFAP (magenta), D) CNPase (magenta), and E) NG2 (magenta). F) Percentages of BrdU phenotypes in the motor cortex at PD 50. Analysis of BrdU+ cell phenotypes revealed that the majority of these cells expressed the glial marker NG2. We found no BrdU+ cells in adult motor cortex in any group that expressed NeuN+, that is, was of a neuronal phenotype. Scale bars = 10 µm for A – E. BrdU, bromodeoxyuridine; NeuN, neuronal nuclei; PD, postnatal day; GFAP, glial fibrillary acidic protein.

Figure 5

BrdU and NG2 co-localizes in the motor cortex of adult rats. A) Confocal image of BrdU (green) and NG2 (magenta) labeling. Arrows indicate co-localization and the cells displayed in B–E). B) NG2 (magenta) labeling, C) BrdU (green) labeling and D) the merged (demonstrating co-localization) image of the two labels. For verification of co-localization, the x-, y-, and z- planes of the confocal image were analyzed by orthogonal view. E) Orthogonal view of BrdU (green) and NG2 (magenta) co-localization. Scale bars = 10 µm. BrdU, bromodeoxyuridine.

Figure 6

A) Alcohol exposure on PD 4–9 did not alter the number of surviving BrdU+ cells at PD80 in the adult motor cortex. There were no significant group differences in the number of BrdU+ cells. PD, postnatal day; SC, suckle control; SI, sham intubated; AE, alcohol-exposed. Data expressed as mean ± SEM. B) Alcohol exposure decreases the number of surviving BrdU+ cells at PD80 relative to PD50 in the adult motor cortex. PD, postnatal day; SI, sham intubated; AE, alcohol-exposed. Data expressed as mean ± SEM. *p<0.05.