In this study, we show that on-going alcohol abuse is associated with a reduced number of Ki67-IR, Sox2-IR, and DCX-IR cells in the human DG. Our data represent a quantitative estimate of proliferating cells, stem/progenitor cells, and immature neurons in the DG from well-characterized human alcoholics with an on-going alcohol abuse and from control subjects. Ki67-IR, Sox2-IR, and DCX-IR were counted in the DG consisting of the GCL and 100 μm of each of the SGZ and the ML. This width of the areas of interest constitutes a compromise between very thin and very wide zones previously examined. Whereas Eriksson et al (1998) have studied the occurrence of BrdU neurons in the SGZ within two neurons width from the GCL, Boldrini et al (2013) counted Ki67 positivities up to several hundreds of μm from the GCL. The reason why investigators have used variable widths when examining SGZ is that this structure is not well defined in the human hippocampus. The CA4 region in primates is much larger than in rodents and the polymorphic layer thins to a narrow SGZ (Amaral et al, 2007), but this zone is still several hundreds of μm wide in humans. When looking for new neurons that may become mature, integrated granule cells, it is reasonable to limit the distance from the GCL in order to avoid counting cells that may have a different fate. In this study we wanted to investigate dividing cells as well as stem/progenitor and immature neurons and therefore decided to set the width to 100 μm from the GCL. We expected this width to allow for identification of positive cells for all three markers in such numbers that possible differences between alcoholics and controls could be discovered. This corresponds approximately to the width of 10 granule cells. The topographical distribution of the markers’ immunoreactivity in the SGZ showed a general reduction in alcoholics compared with controls in all layers (Figure 4). We also observed that Ki67-IR, Sox2-IR, and DCX-IR counts decreased further away outside the 100 μm limit.

Although studies on hippocampal neurogenesis have focused on the SGZ, we decided to also count Ki67-IR, Sox2-IR, and DCX-IR cells in the ML of controls and alcoholics, and found immunoreactivity of all the markers in the ML with a significant reduction in alcoholics in Sox2-IR and DCX-IR, but not in Ki67-IR. These results are somewhat difficult to interpret as the possible involvement of cells in the ML in neurogenesis has not been systematically explored. Many different subtypes of ML interneurons and even migrating developing interneurons are recognized (Amaral et al, 2007; Freund and Buzsaki, 1996; Kohler et al, 2011). Studies in mice have revealed that cell division kinetics in the ML is similar to that of the SGZ (Li et al, 2013) and Sox2 is also expressed in the ML of rats (Hattiangady and Shetty, 2008). In humans, Ki67 and DCX immunoreactivity was also shown in the ML (Freund and Buzsaki, 1996; Han et al, 1993; Ribak and Seress, 1983). DCX-IR cells in ML can alternatively represent astrocytes as observed in both adult hippocampus and human neocortex (Verwer et al, 2007), but still be involved in neurogenesis by assisting in the growth of processes that immature neurons in the SGZ will send out into the ML. In addition, in adult mice, molecular layer perforant pathway (MOPP) cells were demonstrated to innervate newly generated granule neurons (Li et al, 2013). This process most likely involves dendrite sprouting that is linked to DCX expression (Rao and Shetty, 2004). Another possibility is that some of the DCX-IR cells in the ML are immature neurons migrating too far into the ML and not stopping in the GCL, like BrdU-labeled cells may also appear in the ML in experimental animals (Mathews et al, 2010). However, further studies are needed to characterize the cells expressing Sox2 and DCX in the ML in more detail.

To investigate the effects of alcohol abuse on proliferation, we performed immunohistochemistry against Ki67. The density of Ki67-IR cells in the DG, and specifically in the SGZ, was lower in alcoholics as compared with controls. This is in line with animal studies showing that alcohol inhibits hippocampal cell proliferation (Crews et al, 2006a; He et al, 2005) in rodents as heavy binge alcohol does in nonhuman primates (Taffe et al, 2010). Ki67-IR in this study was occasionally colocalized with Sox2-IR that was also significantly reduced in alcoholic DG compared with controls. This finding corroborates the results of studies in nonhuman primates where alcohol treatment reduces the number of cells expressing Ki67 and Sox2 (Taffe et al, 2010).

In rodents, Sox2 is expressed in neural stem cells, neuronal progenitor cells, and astrocytes, and this might imply that the results obtained regarding this marker are due to an additive effect of changes in expression of all cell types expressing Sox2 or because specific cell types are reduced whereas others are unaffected. However, as almost all of the Sox2-IR cells in our study were GFAP negative, we believe that the majority of them rather represent progenitor cells, that is, Type 2a or Type 2b cells in accordance with Knoth et al (2010), who observed that the Sox2-positive cells in the human hippocampus were GFAP negative.

In addition to the reduction among alcoholics of Ki67-IR and Sox2-IR, we found a significant decrease in DCX-IR within the DG and specifically in the SGZ. These results are in line with previous clinically relevant rodent studies of both acute binge and chronic ethanol exposure showing a decrease in DCX-IR (Crews et al, 2006b; He et al, 2005). Most of the DCX cells in the dentate gyrus in our study were also positive for PSA-NCAM (Supplementary Figure S5), further supporting the notion that they possibly represent immature neurons. The coexpression of DCX and PSA-NCAM is in line with a previous study by Ernst et al (2014), who used the same DCX marker and the same IHC protocol (Ernst et al, 2014). It is important to point out that we performed double staining for various markers only in a few cases and our focus rather was to study the effect of alcohol on Ki67-IR, Sox2-IR, and DCX-IR in isolation and therefore used single immunohistochemistry optimized for best signal to noise for each of them.

Furthermore, in this study we found a negative correlation between Ki67 index values and age of subjects in the two groups, whereas Sox2 and DCX index values showed no correlation with age. Other studies in humans have shown a modest decline in neurogenesis during aging (Knoth et al, 2010; Spalding et al, 2013). This difference could be attributed to the limited number of cases, limited age range, and a considerable level of variability between cases. In rodents an exponential decrease takes place during 3–6 months of age to reaches a low and constant level thereafter (Ben Abdallah et al, 2010). In humans, a similar decrease in DCX-IR has been shown (Knoth et al, 2010). The majority of the subjects in our study are older adults, making it difficult to observe minor changes with age.

The discrepancy between cases regarding the expression level of markers could depend on chronicity and magnitude of dosing (Vengeliene et al, 2008). We have been able to obtain information regarding drinking frequency, and degrees of drunkenness from next to kin interviews and/or medical charts, but it is very difficult to obtain reliable information about exact amounts consumed. Extrinsic factors, lifestyles such as exercise, health status, and pharmaceutical drugs are additional factors that may affect neurogenesis and explain some of the variation.

None of the subjects included in this study were reported to be active in sports during the past months, although the degree of daily physical activity was difficult to establish.

Epileptic seizures, known to acutely increase neurogenesis, were not reported by relatives or otherwise documented. Pharmaceutical drugs were detected in the blood of several of the subjects. Three of them had low concentrations of selective serotonin reuptake inhibitors (SSRIs): fluoxetine or sertraline. In a study by Boldrini et al (2009), depressed patients treated with SSRIs showed more Ki67-IR cells in the SGZ than nontreated depressed patients, although this difference was much more prominent in the anterior hippocampus than any region further posteriorly. In the same study they also found that the nontreated patients with depression had almost the same densities of Ki67 as nondepressed controls. As our samples are collected from the mid-portion of hippocampus, we believe that the possible impact of this medication is limited. One subject used methylphenidate and pregabalin. Previous studies have shown variable results regarding effects on proliferation and immature neurons in hippocampus when animals have been exposed to these drugs, and there are no human data on this. In three cases opioids were found; in two of these only at low concentrations and in the third case, the concentrations of the drug (tramadol) and its metabolite strongly suggest an acute accidental overdose and not chronic intake, thus making these findings unlikely to have affected the results.

Development of acute to chronic alcohol abuse involves the transitional stages of tolerance and dependence that may be related to changes in cell types involved in the DG neurogenesis. The persisting addictive desire for alcohol that remains following a protracted period of withdrawal has been suggested to be due to long-lasting structural changes in the hippocampus that affect learning processes and particularly memory of the drug euphoria that drives the maladaptive behaviors to increase the risk of relapse (Fibiger and Phillips, 1988; Hyman and Malenka, 2001). The findings in the present study of reduced densities of markers of stem/progenitor cell subpopulations in alcohol abusers are in line with these anatomical–functional associations and hence the affected cells, particularly the putative neural stem cells, may prove to be an important target in future pharmaceutical interventions.

In summary, our results support the notion that on-going alcohol abuse in humans reduces numbers of proliferating cells, stem/progenitor cells, and immature neurons. The decrements in Sox2-IR were more prominent than those of DCX-IR, suggesting that alcohol primarily causes a depletion of the stem/progenitor cells and that immature neurons are probably secondarily affected. Although the markers we have used may not be specific for the cells participating in the generation of new granule cells, our results are compatible with this possibility, and are in agreement with observations of impaired adult hippocampal neurogenesis by alcohol in animal studies and lend further support for the association between hippocampal dysfunction and alcohol abuse.