Chronic alcohol abuse results in alcohol-related neurodegeneration, and critical gaps in our knowledge hinder therapeutic development. Neural stem cells (NSCs) are a subpopulation of cells within the adult brain that contribute to brain maintenance and recovery. While it is known that alcohol alters NSCs, little is known about how NSC response to alcohol is related to sex, brain region, and stage of differentiation. Understanding these relationships will aid in therapeutic development. Here, we used an inducible transgenic mouse model to track the stages of differentiation of adult endogenous NSCs and observed distinct NSC behaviors in three brain regions (subventricular zone, subgranular zone, and tanycyte layer) after long-term alcohol consumption. Particularly, chronic alcohol consumption profoundly affected the survival of NSCs in the subventricular zone and altered NSC differentiation in all three regions. Significant differences between male and female mice were further discovered.

To address these gaps in knowledge, we utilized recent advances in genetic inducible fate mapping which provides a tool to study longitudinal changes of endogenous NSC populations in the adult brain (). This technology allows us to evaluate differentiation and population changes of endogenous NSCs in response to alcohol. Specifically, we analyzed the effect of chronic alcohol consumption on NSC populations in adult mice and concentrated on the three regions in the mouse brain that are known to contain NSCs: SVZ, SGZ, and TL. In addition, we compared NSC responses in males and females to determine the role of sex in response to alcohol.

The two most commonly studied NSC-containing regions in the adult brain are the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus (). In these regions, alcohol consumption impairs NSC proliferation and neurogenesis (). Recently, the tanycyte layer (TL) of the third ventricle was discovered to contain cells with NSC properties; however, the impact of alcohol on this cell population has not been investigated (). While studies have investigated the effect of alcohol in the SVZ and SGZ, a comparative examination of how NSCs in all three regions (SVZ, SGZ, and TL) respond to alcohol at different stages of differentiation has not been conducted. Despite the broad knowledge that males and females respond differently to alcohol clinically, the role of sex differences in NSC response to alcohol consumption is not well explored. These two unaddressed issues leave substantial gaps in knowledge of how the brain recovers following chronic alcohol abuse.

Several factors affect the response of the brain to alcohol, such as age, sex, duration, and quantity of alcohol consumed (). There are also regional patterns of degeneration and glial pathology observed in patients with a history of chronic alcohol abuse (). As with neurodegenerative diseases, neural damage observed in chronic alcoholism is speculated to be not only caused by neuronal death, but also by inhibition of neurogenic processes (). Thus, the effect of alcohol on neural stem cells (NSCs) has become a topic of interest due to the critical role NSCs play in maintaining neurogenesis throughout life, and possibly in mediating neuro-regeneration after insults ().

Increase in brain cerebrospinal fluid volume is greater in older than in younger alcoholic patients: a replication study and CT/MRI comparison.

Alcohol abuse is the third leading cause of preventable death in the United States, with almost 30% of the population meeting the criteria for alcohol use disorder at some point in life (). It is established that alcohol misuse has detrimental effects on the brain and leads to deficits in cognitive and behavioral function, as well as a condition referred to as alcohol-related neurodegeneration ().

Binge ethanol exposure during adolescence leads to a persistent loss of neurogenesis in the dorsal and ventral hippocampus that is associated with impaired adult cognitive functioning.

Epidemiology of DSM-5 alcohol use disorder: results from the national epidemiological survey on alcohol and related conditions III.

In summary, these data show different behavior of NSCs in the TL compared with both SVZ and SGZ. Long-term alcohol decreased NSCs in males and females, and increased total GFAP + cells without changes of astrogliogenesis in females.

Following long-term ethanol consumption, both males and females exhibited significant decreases of GFPin the TL region by 56% and 40%, respectively ( Figures 7 C and 7D). Interestingly, females had an increase in GFAPcells (75%), whereas males experienced a 67% decrease in GFAPcells ( Figure 7 E). Females did not have a significant reduction in the number of GFAPGFPcells; however, males experienced a 67% decrease in GFAPGFPcells when compared with liquid diet ( Figure 7 F). Neither males nor females had a change in the percentage of GFPcells differentiating into GFAPfollowing long-term alcohol consumption ( Figure 7 G). Individual immunohistochemical stain images can be found in the Supplemental Experimental Procedures Figure S5 ).

The third region we evaluated was the TL of the third ventricle. This region was selected because it has recently been shown to contain cells with NSC potential (). However, in our study we found little evidence of newly differentiating neurons ( Figure S4 ), therefore our primary focus was on GFPand GFAPcell populations. In this region GFAPcells can label astrocytes as well as tanycytes, which are specialized ependymal cells with elongated morphology that share characteristics of astrocytes and radial glia (). The primary role of tanycytes is to communicate chemical signals from the ventricles to the hypothalamus (). Control mice in both standard chow and liquid diet groups had similar numbers of GFP, GFAP, and GFAPGFPcells between both sexes ( Figures 7 A, 7B, and 7D–7F ).

Scale bars, 45 μm, and 15 μm in enlarged images. Values are shown as mean ± SEM, n = 3 mice per sex per group.p < 0.05,p < 0.01,p < 0.001,p < 0.0001 compared with controls, two-way ANOVA with Tukey's test.p < 0.0001 compared with female counterpart, two-way ANOVA with Sidak's test. See also Figures S4 and S5

(H) Schematic representative image of region quantified (bregma from −1.58 to −2.16) and outlined in the red box.

(D–G) Quantification in the TL of average GFP + (D), GFAP + (E), and GFAP + GFP + (F), and the percentage of GFAP + GFP + over total GFP + (G) cells.

(A–C) Representative images of standard chow, liquid diet control, and long-term ethanol male and female mouse brains stained with stem cell marker (GFP green), astrocyte marker (GFAP red), and merged with nuclear maker DAPI (blue). The region in the red box is shown enlarged on the right.

Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus.

In summary, long-term alcohol consumption reduced neurogenesis in both males and females. In addition, females, but not males, had a significant reduction in GFAP + cells.

When assessing SGZ astrogliogenesis, we found no significant differences in GFAP, GFAPGFP, or percentage of GFPcells expressing GFAP between males and females within the control diets ( Figures 6 A, 6B, 6D, and 6E ). In addition, all comparisons between standard chow and control liquid diets were similar ( Figures 6 A, 6B, and 6D–6F). Female mice in the ethanol group had a significant reduction in GFAPcells compared with both standard chow and liquid diet controls; however, males only had a significant reduction relative to the standard chow group ( Figures 6 C and 6D). Long-term ethanol consumption did not have an impact on GFAPGFPpopulations, or the percentage of GFPcells expressing GFAP in males or females ( Figures 6 C, 6E, and 6F). These data suggest that GFAPpopulations in the female SGZ are more sensitive to changes following ethanol consumption compared with males, and that GFAPpopulations respond differently to alcohol in the SGZ compared with the SVZ. Individual immunohistochemical stain images can be found in Figure S3

Scale bars, 45 μm, and 15 μm in enlarged images. Values are shown as mean ± SEM, n = 3 mice per sex per group.p < 0.01,p < 0.001,p < 0.0001, two-way ANOVA with Tukey's test compared with controls. See also Figure S3

(A–C) Representative images of standard chow, liquid diet control, and long-term ethanol male and female mouse brains stained with stem cell marker (GFP green), astrocyte marker (GFAP red), and merged with nuclear maker DAPI (blue). The region in the red box is shown enlarged on the right.

When evaluating SGZ neurogenesis, liquid diet control males and females had similar levels of DCXand DCXGFPcells. Standard chow controls had significantly more DCXcells compared with liquid diet controls ( Figures 5 A, 5B, and 5E). In the long-term ethanol cohort, DCXcells were significantly decreased in males and females when compared with liquid diet controls ( Figures 5 A–5C, and 5E). There was a 49% and 65% decrease in males and females, respectively ( Figure 5 E). There was also a trend of reduction in both males and females in the DCXGFPcell population, albeit statistically insignificant ( Figures 5 C and 5F). Males exhibited a significant reduction (63%) in the percentage of GFPcells labeled with DCX when compared with both standard chow and liquid diet controls, whereas females only had a trending decrease compared with standard chow controls ( Figure 5 G).

Next, we examined the SGZ in the dorsal hippocampus, given its role in cognitive function and active adult neurogenesis (). The standard chow controls had similar numbers of GFPcells; however, the liquid diet controls displayed sex differences, with female mice in the control group having approximately twice the number of GFPcells as their male counterparts ( Figures 5 A–5D ). The SGZ GFPcells also showed a sex-dependent response to ethanol consumption. Alcohol treatment significantly reduced the numbers of GFPcells in both males and females ( Figures 5 C and 5D).

Scale bars, 45 μm, and 15 μm in enlarged images. Values are shown as mean ± SEM, n = 3 mice per sex per group.p < 0.05,p < 0.01,p < 0.0001 compared with controls, two-way ANOVA with Tukey's test.p < 0.05,p < 0.001, compared with other sex counterparts, two-way ANOVA with Sidak's test. See also Figure S3

(H) Schematic image of representative region that was quantified (bregma from −1.58 to −2.16) and outlined in the red box.

(D–G) Quantification in SGZ of average GFP + (D), DCX + (E), and DCX + GFP + (F), and the percentage of DCX + GFP + over total GFP + cells (G).

(A–C) Representative images of standard chow, liquid diet control, and long-term ethanol male and female mouse brains stained with stem cell marker (GFP green), neuronal marker (DCX red), and merged with nuclear maker DAPI (blue). The regions in the red boxes are shown enlarged on the right.

In summary, these data show the NSCs in the SVZ are sensitive to long-term ethanol consumption, more so in females. Chronic ethanol consumption altered neurogenesis by inducing a drastic loss of DCX + GFP + neuroblasts/immature neurons in the oRMS, and a subsequent loss of NeuN + GFP + in the olfactory bulb. Also, alcohol consumption decreased the survival of newly differentiated astrocytes (GFAP + GFP + ).

We further assessed the effect of ethanol consumption on NSC astroglial differentiation by using glial fibrillary acidic protein (GFAP) as a glial marker. In the SVZ region of the control mice, the majority of GFPcells were co-labeled with GFAP ( Figures 4 A–4C ). To determine astrogliogenesis from the SVZ NSCs expressing GFAP, we quantified those GFP and GFAP co-labeled cells in the oRMS (indicated by the red box) (). No significant differences were found in the number of GFAPcells between the standard chow and control liquid diets; however, females in the liquid diet control group had significantly less GFAPcells compared with their male counterparts ( Figures 4 A–4D). In ethanol-treated mice, males showed a trend of reduction in GFAPcells, albeit statistically insignificant, compared with both standard chow and control liquid diets ( Figures 4 C and 4D). GFAPGFPcells decreased by 90% and 97% in males and females, respectively ( Figures 4 C and 4E). Furthermore, the percent of GFPcells expressing GFAPwas significantly reduced in females (92%) and had a trend of reduction in males ( Figure 4 F). Ethanol-treated females had a significantly lower percentage of GFPcells expressing GFAPcompared with their male counterparts. Individual immunohistochemical stain images can be found in the Supplemental Experimental Procedures Figure S2 ).

Scale bars, 45 μm, and 15 μm in enlarged images. Values are shown as mean ± SEM, n = 3 mice per sex per group.p < 0.05,p < 0.01,p < 0.0001 compared with controls, two-way ANOVA with Tukey's test.p < 0.05,p < 0.001,p < 0.0001 compared with other sex counterparts, two-way ANOVA with Sidak's test. See also Figure S2

(D–F) Quantification of average GFAP + (D) and GFAP + GFP + (E), and the percentage of GFAP + GFP + over total GFP + cells (F).

(A–C) Representative images of standard chow, liquid diet control, and long-term ethanol male and female mouse brains stained with stem cell marker (GFP green), astrocyte marker (GFAP red), and merged with nuclear maker DAPI (blue). The region in the red box is shown enlarged at the bottom.

Glial fibrillary acidic protein-expressing neural progenitors give rise to immature neurons via early intermediate progenitors expressing both glial fibrillary acidic protein and neuronal markers in the adult hippocampus.

Next we examined the neurogenic capacity of SVZ NSCs after ethanol consumption by using the doublecortin (DCX) antibody, a marker of newly differentiated neuroblasts and immature neurons (). Males and females receiving the liquid diet had fewer DCXand DCXGFPcells in the oRMS compared with standard chow controls ( Figures 2 A–2C, 2F, and 2G). In the liquid diet controls, DCXGFPaccounted for 35% and 22% of the total GFP cells in control males and females, respectively ( Figures 2 B and 2G). Long-term ethanol consumption significantly decreased DCXexpression by 54% in males and 66% in females ( Figures 2 C and 2F). In addition, DCXGFPcells were reduced by 82% in males and 87% in females ( Figures 2 C and 2G). Males but not females demonstrated a reduction in the percent of GFPcells becoming DCXfollowing long-term ethanol consumption ( Figure 2 H). The greater loss of DCXGFPcells ( Figure 2 G) than that of the total DCXcells indicates that recently differentiated NSCs are more susceptible to ethanol-mediated toxicity than older differentiated cells. To further confirm the effect of chronic ethanol consumption on neurogenesis of NSCs, dual immunolabeling with GFP and NeuN (a mature neuronal marker) was conducted in the olfactory bulb. As shown in Figure 3 , chronic ethanol treatment significantly reduced the numbers of newly generated mature neurons (NeuNGFP) and GFPcells in the olfactory bulb, exhibiting a trend similar to DCXGFPin the oRMS ( Figure 3 ).

Properties of doublecortin-(DCX)-expressing cells in the piriform cortex compared to the neurogenic dentate gyrus of adult mice.

We focused on the rostral lateral ventricle, which harbors endogenous NSCs and is an area of active neurogenesis in adult mammalian brains (). The Nestin-CreER;R26R-YFP transgenic mice allowed us to trace the NSCs that were present at the time of tamoxifen injection by their expression of yellow fluorescent protein (YFP) (). Tamoxifen, administered at 2 months of age, enabled expression of the YFP reporter gene in Nestin-expressing NSCs in adult mice. The YFP signal was further enhanced by immunohistochemistry using a GFP antibody. Since the YFP reporter gene was activated by tamoxifen, only cells expressing Nestin at the time of tamoxifen injection would be labeled with YFP. Since YFP is constitutively active, all progeny cells from that induced population will also express YFP, regardless of differentiation or division. This provides the capacity to study a population of NSCs and their progeny in response to alcohol consumption. This genetic tracing technique allowed us to examine three phases of NSCs: NSCs (GFP), newly differentiated NSCs (DCXGFPor GFAPGFP), and neurons or astrocytes that were present before tamoxifen induction (DCXor GFAP). Cells that were double-labeled with GFP and a marker of differentiation (DCX or GFAP) represent newly differentiated cells after tamoxifen induction of the YFP expression in Nestincells. Cells that differentiated prior to tamoxifen induction and no longer expressed Nestin would not be co-labeled with GFP. In standard chow controls, there were more DCXand DCXGFP, and fewer GFAPGFPcells, indicating that the liquid diet itself may influence cell proliferation () and probably neurogenesis ( Figures 2 and 3 ), likely due to the high fat in the liquid diet (). Thus, ethanol treatment was compared with liquid diet controls. Male and female mice on control diets had comparable numbers of GFPNSCs in the SVZ region ( Figures 2 A and 2B). Mice receiving the ethanol diet exhibited significant alterations of SVZ NSCs in a temporal- and sex-dependent manner. In the SVZ region (indicated by orange lines), long-term treatment decreased GFPcells by 98% in females and 89% in males ( Figures 2 A–2D). The total number of GFPcells was reduced by 94% in females and 74% in males in the origin of the rostral migratory stream (oRMS) (indicated by the red box) ( Figures 2 A–2C and 2E).

Values are shown as mean ± SEM, ∗ p < 0.05 compared with control, # p < 0.05 compared with male counterpart, two-way ANOVA with Tukey's test, n = 3 mice per sex per group.

(A and B) Representative images of control and long-term ethanol male and female mouse brains stained with stem cell marker (GFP green), mature neuronal marker (NeuN red), and merged with nuclear maker DAPI (blue). Scale bars, 32 μm. n = 3 mice per sex per group.

Values are shown as mean ± SEM, n = 3 mice per sex per group.p < 0.05,p < 0.001,p < 0.0001 compared with controls, two-way ANOVA with Tukey's test.p < 0.05 compared with male counterpart, two-way ANOVA with Sidak's test. Scale bars, 45 μm, and 15 μm in enlarged images. See also Figure S2

(I) Schematic image of representative region quantified (bregma from 0.5 to 1.08) and outlined in the red box.

(D–H) Quantification of average total GFP + cells in SVZ and origin of the rostral migratory stream (D), GFP + within the SVZ (E), DCX + (F), and DCX + GFP + (G), and the percentage of DCX + GFP + over total GFP + cells (H).

(A–C) Representative images of standard chow (Ctrl SC), liquid diet control (Ctrl LD), and long-term ethanol (LT EtOH) male and female mouse brains stained with stem cell marker (GFP green), neuronal marker (DCX red), and merged with nuclear maker DAPI (blue). The regions in the red boxes are shown enlarged at the bottom.

Similar to humans, mice showed individual variations in response to alcohol intake, ranging from ataxia to loss of righting reflex. To evaluate ethanol intoxication, an ordinal scale was created based upon home cage behaviors ranging from asymptomatic to severe on a scale of 0–4, respectively ( Figure 1 H). These behaviors have previously been associated with intoxication in mice (). All animals in the ethanol group tolerated the 2-week ramping stage without abnormal behaviors. Among 25 mice fed with 4% ethanol, 4 males (30.8%) and 4 females (33.3%) were kept on a sustained 4% ethanol diet for 28 days with mild symptoms and thus were included in the “long-term” study. On the other hand, 9 males (69.2%) and 8 females (66.7%) showed severe symptoms of intoxication reaching a score of 4 between 6 and 24 days with 4% ethanol. Five males and 4 females died between 6 and 24 days of 4% ethanol, and were not included in this study. Other mice with severe symptoms were euthanized between 6 and 11 days and not included for further histopathological analyses in this study. Ethanol females displayed more severe symptoms of intoxication and escalated in severity at a higher rate than males (p = 0.009) ( Figure 1 I). Blood alcohol analysis showed a wide range of blood alcohol concentrations (BACs) and blood acetaldehyde concentrations, which did not correlate with behavioral severity for either sex ( Figure S1 ). One source of variability may be due to blood samples collected in the morning, while mice typically feed in the first few hours of the dark cycle. In addition, since mice have unrestricted access to the diet it is possible that mice with higher BACs consumed the diet more recently than mice with lower BACs.

Adult Nestin-CreER:R26R-YFP bitransgenic mice were used to trace the fate of endogenous NSCs following tamoxifen induction, and chronic ethanol feeding with the Lieber-DeCarli ethanol liquid diet ( Figure 1 A). Both pair-fed control and ethanol-fed mice were given ad libitum access to their respective diets and water. To ensure that both ethanol and control mice received diets with the same caloric value, maltose-dextrin was used to calorie balance the diets. At the start of the study, males weighed more than females; however, there was no significant difference between the body weights of control or ethanol groups ( Figures 1 B and 1C). Male mice in both groups had a higher average daily diet consumption compared with their female counterparts. Control males consumed 5% more diet than control females, and ethanol males consumed 7% more than ethanol females ( Figure 1 D). Due to the greater consumption, male mice consumed more grams of ethanol compared with females; however, females consumed 13% more grams of ethanol per kilogram of body weight ( Figures 1 E and 1F). There were no significant changes in body weight gains, but mice in the ethanol group tended to have less weight gain ( Figure 1 G).

Values are shown as mean ± SEM,p < 0.05 compared with control,p < 0.05 compared with other sex in the same group, male mice n = 13, female mice n = 12; two-way ANOVA with Tukey's test. See also Figure S1

(I) Graph comparing the severity of alcohol-induced symptoms over time. Red line, female; blue line, male; shadow, error bars. Values are medians in each day evaluated by a random slope model.

Discussion

Geil et al., 2014 Geil C.R.

Hayes D.M.

McClain J.A.

Liput D.J.

Marshall S.A.

Chen K.Y.

Nixon K. Alcohol and adult hippocampal neurogenesis: promiscuous drug, wanton effects. Stangl and Thuret, 2009 Stangl D.

Thuret S. Impact of diet on adult hippocampal neurogenesis. Our study is to employ a genetic inducible fate mapping model to study the effects of chronic alcohol intake on adult brain NSCs and their progeny, comparatively, in three main brain regions in both male and female mice. A particular interesting finding is the observation of changes in the hypothalamic NSCs in response to alcohol consumption. One limitation in this study was the high mortality of mice, likely due to the administration of ethanol since mice in the ethanol group were not given a choice of control diet. However, this model eliminates the variables in other models, such as ethanol dose and caloric intake that also impact NSCs ().

+ cells resulted from a combination of NSC astrogliogenesis and astrocyte reactivation, and varied among brain regions. Previous studies evaluated regional and durational differences in GFAP expression ( Franke, 1995 Franke H. Influence of chronic alcohol treatment on the GFAP-immunoreactivity in astrocytes of the hippocampus in rats. Franke et al., 1997 Franke H.

Kittner H.

Berger P.

Wirkner K.

Schramek J. The reaction of astrocytes and neurons in the hippocampus of adult rats during chronic ethanol treatment and correlations to behavioral impairment. Dalcik et al., 2009 Dalcik H.

Yardimoglu M.

Fiiz S.

Gonca S. Chronic ethanol-induced glial fibrillary acidic protein (GFAP) immunoreactivity: an immunocytochemical observation in various regions of adult rat brain. Dalcik et al., 2009 Dalcik H.

Yardimoglu M.

Fiiz S.

Gonca S. Chronic ethanol-induced glial fibrillary acidic protein (GFAP) immunoreactivity: an immunocytochemical observation in various regions of adult rat brain. Kane et al., 2014 Kane C.J.

Phelan K.D.

Douglas J.C.

Wagoner G.

Johnson J.W.

Xu J.

Phelan P.S.

Drew P.D. Effects of ethanol on immune response in the brain: region-specific changes in adolescent versus adult mice. Franke, 1995 Franke H. Influence of chronic alcohol treatment on the GFAP-immunoreactivity in astrocytes of the hippocampus in rats. Franke et al., 1997 Franke H.

Kittner H.

Berger P.

Wirkner K.

Schramek J. The reaction of astrocytes and neurons in the hippocampus of adult rats during chronic ethanol treatment and correlations to behavioral impairment. In terms of astrocytes, we observed regional and sex differences in astrocytic responses following alcohol consumption. The net changes of GFAPcells resulted from a combination of NSC astrogliogenesis and astrocyte reactivation, and varied among brain regions. Previous studies evaluated regional and durational differences in GFAP expression (); however, our study further examined both variables together with sex differences in adult astrocyte response to alcohol consumption. In addition, using the genetic induced fate mapping technology, we were able to distinguish the newly NSC-differentiated astrocytes from the reactivated astrocytes. Some studies reported increases in GFAP immuno-labeled cells in the hippocampus following alcohol consumption (). In contrast, we found no alteration of GFAP expression in males, but decreased GFAP in female hippocampal dentate gyrus. Using a similar dosing paradigm, and in agreement with us, Franke and colleagues also observed similar GFAP responses (). These suggest that both ethanol dosing regimen and sex play key roles in astrocytic responses.

+), newly differentiated immature (GFP+DCX+) and mature (GFP+NeuN+) neurons, and immature neurons (GFP−NeuN+) that were present before tamoxifen induction. In the SVZ, we discovered that NSCs lining the ventricle are most susceptible to ethanol, followed by newly differentiated NSCs, and, finally, cells that had differentiated before the onset of the study. This subtle, yet important, distinction between the stages of NSC differentiation is crucial in developing an understanding of how ethanol progressively impacts the adult brain. Furthermore, our data also suggested the potential mechanisms underlying alcohol-induced neurogenesis deficits in SVZ, including a reduced NSC pool, inhibition of neuronal differentiation, and/or reduced migration of newly differentiated neurons. The latter has also been well documented in rodents treated with alcohol during neural development ( Miller, 1993 Miller M.W. Migration of cortical neurons is altered by gestational exposure to ethanol. One distinguishing aspect of our study is that using a genetic inducible fate tracing mouse model enabled us to identify three distinct phases contributing to neurogenesis and evaluate the unique susceptibility of each phase to ethanol. The three phases are NSCs (GFP), newly differentiated immature (GFPDCX) and mature (GFPNeuN) neurons, and immature neurons (GFPNeuN) that were present before tamoxifen induction. In the SVZ, we discovered that NSCs lining the ventricle are most susceptible to ethanol, followed by newly differentiated NSCs, and, finally, cells that had differentiated before the onset of the study. This subtle, yet important, distinction between the stages of NSC differentiation is crucial in developing an understanding of how ethanol progressively impacts the adult brain. Furthermore, our data also suggested the potential mechanisms underlying alcohol-induced neurogenesis deficits in SVZ, including a reduced NSC pool, inhibition of neuronal differentiation, and/or reduced migration of newly differentiated neurons. The latter has also been well documented in rodents treated with alcohol during neural development ().

+ NSCs, and particularly the absence of GFP labeling in regions lining the lateral ventricle, suggested cytotoxicity as a mechanism. However, it is possible that inhibition of proliferation also contributed to the reduction of GFP+ NSCs, which has been reported in previous studies ( Golub et al., 2015 Golub H.M.

Zhou Q.G.

Zucker H.

McMullen M.R.

Kokiko-Cochran O.N.

Ro E.J.

Nagy L.E.

Suh H. Chronic alcohol exposure is associated with decreased neurogenesis, aberrant integration of newborn neurons, and cognitive dysfunction in female mice. Hansson et al., 2010 Hansson A.C.

Nixon K.

Rimondini R.

Damadzic R.

Sommer W.H.

Eskay R.

Crews F.T.

Heilig M. Long-term suppression of forebrain neurogenesis and loss of neuronal progenitor cells following prolonged alcohol dependence in rats. Nixon and Crews, 2002 Nixon K.

Crews F.T. Binge ethanol exposure decreases neurogenesis in adult rat hippocampus. Zappaterra and Lehtinen, 2012 Zappaterra M.W.

Lehtinen M.K. The cerebrospinal fluid: regulator of neurogenesis, behavior, and beyond. + cells in the region surrounding the SVZ during a short-term ethanol treatment (our unpublished data). One reason may be that the insult of ethanol and metabolites on the SVZ initiates an injury response leading to local astrocyte reactivation ( Anderson et al., 2014 Anderson M.A.

Ao Y.

Sofroniew M.V. Heterogeneity of reactive astrocytes. Brahmachari et al., 2006 Brahmachari S.

Fung Y.K.

Pahan K. Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide. Sutherland et al., 2013 Sutherland G.T.

Sheahan P.J.

Matthews J.

Dennis C.V.

Sheedy D.S.

McCrossin T.

Curtis M.A.

Kril J.J. The effects of chronic alcoholism on cell proliferation in the human brain. We found that NSCs lining the SVZ of the rostral lateral ventricles were most susceptible to the effects of ethanol. The significant reduction in the number of GFPNSCs, and particularly the absence of GFP labeling in regions lining the lateral ventricle, suggested cytotoxicity as a mechanism. However, it is possible that inhibition of proliferation also contributed to the reduction of GFPNSCs, which has been reported in previous studies (). One explanation for this heightened effect is the proximity of SVZ NSCs to the cerebrospinal fluid. It has previously been shown that cerebrospinal fluid can directly influence cell behavior and neurogenesis (). Direct exposure to ethanol and other toxic metabolites of ethanol, such as acetaldehyde, may contribute to amplified sensitivity of this population. There was a robust increase in GFAPcells in the region surrounding the SVZ during a short-term ethanol treatment (our unpublished data). One reason may be that the insult of ethanol and metabolites on the SVZ initiates an injury response leading to local astrocyte reactivation (). The mechanism and extent to which astrocyte reactivation contributes to changes in SVZ neurogenesis presents a unique opportunity for future studies. On the other hand, a previous study did not detect significant reduction of SVZ cell proliferation in the postmortem brains of a small cohort of chronic alcoholic patients (). Although this needs to be further confirmed with more human brains, it calls for animal models of chronic alcohol abuse that more closely mimic what occurs in humans.

Robins et al., 2013 Robins S.C.

Stewart I.

McNay D.E.

Taylor V.

Giachino C.

Goetz M.

Ninkovic J.

Briancon N.

Maratos-Flier E.

Flier J.S.

et al. alpha-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors. Haan et al., 2013 Haan N.

Goodman T.

Najdi-Samiei A.

Stratford C.M.

Rice R.

El A.E.

Bellusci S.

Hajihosseini M.K. Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus. Rodriguez et al., 2005 Rodriguez E.M.

Blazquez J.L.

Pastor F.E.

Pelaez B.

Pena P.

Peruzzo B.

Amat P. Hypothalamic tanycytes: a key component of brain-endocrine interaction. Robins et al., 2013 Robins S.C.

Stewart I.

McNay D.E.

Taylor V.

Giachino C.

Goetz M.

Ninkovic J.

Briancon N.

Maratos-Flier E.

Flier J.S.

et al. alpha-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors. Rodriguez et al., 2005 Rodriguez E.M.

Blazquez J.L.

Pastor F.E.

Pelaez B.

Pena P.

Peruzzo B.

Amat P. Hypothalamic tanycytes: a key component of brain-endocrine interaction. Robins et al. (2013) Robins S.C.

Stewart I.

McNay D.E.

Taylor V.

Giachino C.

Goetz M.

Ninkovic J.

Briancon N.

Maratos-Flier E.

Flier J.S.

et al. alpha-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors. + cells in the α-1 tanycyte region as well. This suggests that α-1 tanycytes may also have some NSC properties. Further studies should be conducted to verify this finding. The TL is an intriguing region, given its novelty in the NSC field as well as its relationship with the surrounding brain regions such as the dorsomedial hypothalamus, paraventricular nucleus, and arcuate nucleus (). Tanycytes are the primary cells found lining the TL of the third ventricle, and their main function is to relay signals from the CSF to the surrounding regions that are critical for regulating functions such as feeding, sleep, and water balance ().showed that dorsal α-2 tanycytes present along the third ventricle in the region of the arcuate nucleus were unique in their possession of NSC-like potential. However, in our study, we observed GFPcells in the α-1 tanycyte region as well. This suggests that α-1 tanycytes may also have some NSC properties. Further studies should be conducted to verify this finding.

McNay et al., 2012 McNay D.E.

Briancon N.

Kokoeva M.V.

Maratos-Flier E.

Flier J.S. Remodeling of the arcuate nucleus energy-balance circuit is inhibited in obese mice. Haan et al., 2013 Haan N.

Goodman T.

Najdi-Samiei A.

Stratford C.M.

Rice R.

El A.E.

Bellusci S.

Hajihosseini M.K. Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus. + staining and no changes in response to ethanol consumption. In the previous studies evaluating neurogenesis in this region, neuronal marker NeuN was used, which may be one explanation for the discrepant findings in our study ( McNay et al., 2012 McNay D.E.

Briancon N.

Kokoeva M.V.

Maratos-Flier E.

Flier J.S. Remodeling of the arcuate nucleus energy-balance circuit is inhibited in obese mice. Haan et al., 2013 Haan N.

Goodman T.

Najdi-Samiei A.

Stratford C.M.

Rice R.

El A.E.

Bellusci S.

Hajihosseini M.K. Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus. Rojczyk-Golebiewska et al., 2014 Rojczyk-Golebiewska E.

Palasz A.

Wiaderkiewicz R. Hypothalamic subependymal niche: a novel site of the adult neurogenesis. While recent evidence has indicated that there may be active adult neurogenesis in the TL (), we saw very little DCXstaining and no changes in response to ethanol consumption. In the previous studies evaluating neurogenesis in this region, neuronal marker NeuN was used, which may be one explanation for the discrepant findings in our study (). NeuN labels more mature neurons, whereas DCX labels immature migratory neurons. Alternatively, the discrepancy in finding of TL neurogenesis in hypothalamus may be attributed to different animal age, i.e., 18-week-old mice in our study versus 8- to 12-week-old mice in others (). Further investigation with multiple neuronal markers in animals with a larger age range would be beneficial to elucidate the presence and degree of neurogenesis in this region.

+ cells in the TL were quite interesting. Long-term alcohol consumption resulted in a decrease in NSCs, but not as dramatic of a decrease as was observed in the SVZ. Since NSCs in the TL line the third ventricle, we expected that their behavior would be similar to NSCs in the SVZ of the lateral ventricle; however, this was not observed in the present study. Increases of GFAP+ cells were in females following alcohol consumption. Given the role that tanycytes play in communication with the hypothalamus and paraventricular nucleus in regulation of feeding behavior and other endocrine-related functions, further studies are necessary to better understand the effect of alcohol on this cell population ( Rodriguez et al., 2005 Rodriguez E.M.

Blazquez J.L.

Pastor F.E.

Pelaez B.

Pena P.

Peruzzo B.

Amat P. Hypothalamic tanycytes: a key component of brain-endocrine interaction. While we did not observe substantial neurogenesis, the changes in NSCs and GFAPcells in the TL were quite interesting. Long-term alcohol consumption resulted in a decrease in NSCs, but not as dramatic of a decrease as was observed in the SVZ. Since NSCs in the TL line the third ventricle, we expected that their behavior would be similar to NSCs in the SVZ of the lateral ventricle; however, this was not observed in the present study. Increases of GFAPcells were in females following alcohol consumption. Given the role that tanycytes play in communication with the hypothalamus and paraventricular nucleus in regulation of feeding behavior and other endocrine-related functions, further studies are necessary to better understand the effect of alcohol on this cell population (). Since the morphology of the cells in our study look similar to tanycytes, and that GFAP and Nestin are expressed in tanycytes, further studies are needed to fully validate the identity of this cell population.