Impact of alcohol and drug abuse on hippocampal neurogenesis in humans

Sammanfattning: Hippocampus is one of the few brain regions in which adult neurogenesis is known to occur. Adult neurogenesis in the hippocampus is considered to be important for higher cognitive function, most notably in memory processes and mood regulation. Alcohol abusers are often diagnosed with memory dysfunction. Several animal studies have reported impairment of alcohol on adult neurogenesis in hippocampus, however in studies of adult human alcohol abusers, no conclusive results have been obtained so far. The work presented in this thesis focuses on understanding the effect of alcohol on hippocampal neurogenesis and is based on studies of carefully phenotyped postmortem human subjects. The studies include the effect of chronic abuse of alcohol on hippocampal granule cells, analyzed using stereological principles and determination of the densities of neural stem/progenitor cells and immature neurons in dentate gyrus. Further, hippocampal cell turnover was studied regarding the effect of alcohol and cocaine using retrospective bomb-pulse derived carbon-14 birth dating procedure. In Paper I we assessed the effect of alcohol on proliferating cells, stem/progenitor cells and immature neurons using immunohistochemistry with antibodies against Ki67, Sox2 and DCX in the subgranular zone and other layers of the dentate gyrus of the hippocampus. All subjects included in this study had an on-going alcohol abuse for at least four weeks prior to death, a time period that was chosen based on data about the time that elapse from that a neural stem cell after asymmetric division becomes an integrated neuron in the granule cell layer of the dentate gyrus. We found that alcohol negatively affect the proliferating cells, stem/progenitor cells and immature neurons in the hippocampus. The effect was found to be prominent in the subgranular zone and evenly distributed across the distances from the granular cell layer. Alcohol has more pronounced effect on Sox2-IR cells than DCX-IR cells which suggest that the stem/progenitor cells are primarily targeted and that the immature neurons are secondarily affected. In Paper II we investigated whether the effect of alcohol on neurogenesis might over time affect the number and density of granule cells in the hippocampus. A morphometric method based on the principle of optical fractionator of stereology was applied on blocks of hippocampus at a standardized anatomical location. Neuronal nuclear antigen NeuN was used as a marker for mature granule cell in this study. We found that alcohol significantly reduces the total number and density of granule cells in addition to a decrease in the volume of GCL in hippocampus. We also report that the significant difference in density was primarily due to a reduction of granule cell number. There are substantial inter-individual differences in granule cell numbers, and alcohol seemingly has a stronger impact on this than age of the subjects. In Paper III we studied the difference in neuronal and non-neuronal cell turnover rate in control, chronic alcohol abusers and cocaine abusers. We have used a retrospective 14C birth dating procedure to estimate the average age of hippocampal cell populations and mathematical modeling to calculate the turnover rate of hippocampal cells. The turnover rate of both neuronal and non-neuronal cells in cocaine abusers were indistinguishable from control subjects, whereas we observed a lower turnover rate in alcohol abusers compared to controls. However, this difference was not statistically significant when the results were corrected for the age of the subjects. Due to an increased loss of hippocampal neurons in alcoholics, it cannot be excluded that the true turnover rates may be lower in this group. In conclusion, in this thesis, we have found support for impairment of neurogenesis in the hippocampus in alcoholics and that alcoholics over a lifetime have lost a substantial portion of their granule cells, which may be explained by both a reduced addition of new cells to the dentate gyrus and an increased removal of cells. Using 14C analysis of neuronal nuclei we could not detect a significant difference in turnover of granule cells between alcoholics and controls. A mathematical modeling considering the effect of cell loss in alcoholics and/or a separate analysis of 14C in the granule cell population would be desirable to more in detail understand the dynamics.