Long-term suppression of forebrain neurogenesis and loss of neuronal progenitor cells following prolonged alcohol dependence in rats.

Hippocampal cell proliferation during protracted abstinence. (a) Top: schematic illustration of the sampled area (grey outline) for BrdU-immunoreactive (IR) cell counting of the dentate gyrus subgranular zone (SGZ) in a coronal rat section (adapted from Vaidya et al. 2007). Bottom: data showing the number of BrdU-IR positive cells/mm2 (mean±S.E.M.) in the SGZ at various time-points after the last intoxication cycle (■) and in alcohol-naive controls (□, n=16). The number of BrdU-positive cells varied as a function of time (p<0.0001), and was decreased immediately after alcohol exposure (day 0, n=9), followed by a rebound burst on day 3 (n=8), returning to normal levels on day 7 (n=8) and day 21 (n=3). (b) Illustrative brightfield photomicrographs showing clusters of BrdU-positive cells in the SGZ at the different time-intervals. Scale bar, 30 μm; Bregma level=−1.8 to −5.6 mm according to Paxinos & Watson (2005). (c) Data showing the number of Ki67-IR positive cells/mm2 (mean±S.E.M.) at the respective time-point after the last intoxication cycle [■, n=9 (day 0), n=8 (day 3), n=8 (day 7), n=3 (day 21)] and in alcohol-naive controls (□, n=16). ** p<0.01, *** p<0.001 vs. controls. (For detailed statistics, see Results section.)

Hippocampal neurogenesis during protracted abstinence. (a) Data showing the number of newly differentiated, doublecortin-immunoreactive (DCX-IR) cells/mm2 (mean±S.E.M.) in the subgranular zone (SGZ) at varying intervals after the last intoxication cycle (■) and alcohol-naive controls (□, n=16). The number of positive cells varied as a function of time (p<0.0001). DCX-IR cells were markedly decreased immediately following exposure (day 0, n=9), showed a trend level rebound (day 3, n=8), and then returned to levels that did not differ from controls (day 7, n=8; day 21, n=3). *** p<0.001 vs. controls. (For detailed statistics, see Results section.) (b) Illustrative brightfield photomicrographs of DCX-IR-labelled cells within the SGZ at the different time-intervals. Scale bar, 30 μm; Bregma level=−1.8 to −5.6 mm.

Cell proliferation in the subventricular zone (SVZ) during protracted abstinence. (a) Top: schematic illustration representing the sampled areas (■) within the SVZ (grey outline) for densitometric evaluation of 5′-bromo-2-deoxyuridine-immunoreactivity (BrdU-IR) in coronal rat sections (adapted from Vaidya et al. 2007). Bottom: data showing mean integrated optical densities (IOD; mean±S.E.M.) of BrdU-IR-positive cells in relation to alcohol-naive controls (□, n=16) at different time-points after the last intoxication cycle (■). BrdU-IR varied as a function of time (p<0.001). BrdU-IR cells were markedly suppressed immediately following exposure (day 0, n=8), followed by a rebound burst on day 3 (n=8), and finally a return to levels on day 7 (n=8) and 21 (n=3) that were numerically lower than controls, although the individual post-hoc comparisons failed to reach significance. ** p<0.01 vs. controls. (For detailed statistics, see Results section.) (b) Illustrative brightfield photomicrographs showing clusters of BrdU-IR-positive proliferating cells in the SVZ at the different time-points. Scale bar, 80 μm; Bregma level=+1.0 to +0.2 mm. (c) Data showing mean integrated optical densities (IOD; mean±S.E.M.) from Ki67-IR at various time-points after the last intoxication cycle (■, n=9 (day 0), n=8 (day 3), n=8 (day 7), n=3 (day 21)] and in alcohol-naive controls (□, n=16).

Neurogenesis in the subventricular zone (SVZ) during protracted abstinence. (a) Data showing doublecortin-immunoreactivity (DCX-IR) as normalized mean integrated optical density (IOD; mean±S.E.M.) in the SVZ at varying intervals after the last intoxication cycle (■) and in controls (□; absolute value 306±46, n=15). DCX-IR varied as a function of time (p<0.00001). It was suppressed immediately following exposure (day 0, n=8), rebounded to elevated levels on day 3 (n=8) and day 7 (n=8), and was again suppressed on day 21 (n=3). *** p<0.001 vs. controls. (For detailed statistics, see Results section.) (b) Illustrative brightfield photomicrographs of SVZ DCX-IR neurons at varying intervals following exposure. Scale bar, 80 μm; Bregma level=+1.0 to +0.2 mm.

Loss of SOX2-immunoreactive (IR)-labelled neural stem cells in the subventricular zone (SVZ) during protracted abstinence. (a) Data show mean integrated optical densities (IOD; mean±S.E.M.) of SOX2-IR-labelled neural stem cells at varying intervals following the last intoxication cycle (■) compared to control rats (□, n=16). SOX2-IR varied as a function of time (p<0.0001). Levels did not differ from controls on day 0 (n=8) and 3 (n=8), but were suppressed on day 7 (n=8), and remained suppressed on day 21 (n=3). ** p<0.01, *** p<0.001. (For detailed statistics, see Results section). (b) Illustrative brightfield photomicrographs of SOX2-IR-labelled neural stem cells in the SVZ at the different time-points. Scale bar, 80 μm; Bregma level=+1.0 to +0.