The influence of sex on hippocampal neurogenesis and neurotrophic responses on the persistent effects of adolescent intermittent ethanol exposure into adulthood

Abstract

In the United States, approximately 90% of alcohol consumed by adolescents is binge drinking. Binge-like ethanol exposure during adolescence promotes dysregulation of neurotrophic responses and neurogenesis in the hippocampus. These effects include changes in proliferation, regulation, differentiation, and maturation of neurons, and there is indication that such effects may be disproportionate between sexes. This study determined whether sex impacts neurotrophic responses and neurogenesis in adulthood after adolescent intermittent ethanol (AIE) exposure. To determine this, adolescent rats underwent AIE with ethanol (5 g/kg). In adulthood, animals were euthanized, and immunohistochemical techniques and ELISAs were utilized to determine AIE effects on sex-specific neurogenesis factors and neurotrophic markers, respectively. AIE exposure led to a significant decrease in neurogenesis in the dentate gyrus of the hippocampal formation indicated by reductions in the numbers of DCX+, SOX2+ and Ki-67+ cells in male and female AIE-exposed rats. Additionally, AIE increased markers for the pro-inflammatory cytokines, TNF-α and IL-1β, in the hippocampus into adulthood in male AIE-exposed rats only. No significant AIE-induced differences were observed in the anti-inflammatory cytokines, IL-10 and TGF-β, nor in the neurotrophic factors BDNF and GDNF. Altogether, our findings indicate that although AIE did not have a persistent effect on hippocampal neurotrophic levels, there was still a reduction in neurogenesis. The neurogenic impairment was not sex specific, but the neurogenic deficits in males may be attributed to an increase in pro-inflammatory cytokine expression. A persistent impairment in neurogenesis may have an impact on both behavioral maladaptations and neurodegeneration in adulthood.

Introduction

Alcohol use disorders (AUDs) are a persistent public health challenge. According to the Centers for Disease Control and Prevention (CDC) excessive alcohol use accounts for more than 380 deaths each day in the United States, consequentially making it the 4^th leading cause of preventable deaths (Gonzales K et al., 2014). Among this affected population adolescences are the most susceptible to alcohol-related deaths (Bonnie RJ OCM, 2004). Adolescence is the transitional period from puberty to adulthood that involves maturation in both a physical and psychological context. Unfortunately, during adolescence individuals are also apt to face social and developmental pressures that often lead to the use of recreational drugs, the most common being alcohol (Gray KM and Squeglia LM, 2018;Johnston L, O’Malley, PM, Miech, RA, Bachman, JG & Schulenberg JE 2017;Johnston LD MR, O’Malley PM, Bachman JG, Schulenberg JE, & Patrick ME, 2018;Teunissen HA et al., 2012). Among United States high school students, 29.2% reported current alcohol use compared to 21.7% who reported marijuana use (Jones CM CH, Deputy NP, et al. , 2020). Unlike adults 21 and older who have legal access to alcohol, adolescents tend to drink in a more intermittent but binge-like pattern when alcohol is accessible (Hargreaves GA et al., 2009). In 2018, 14% of 12^th grade high school students reported binge drinking in the previous two weeks (Johnston LD MR, O’Malley PM, Bachman JG, Schulenberg JE, & Patrick ME, 2018). Binge pattern drinking produces multiple liabilities for adolescents. For example, during adolescence, neural plasticity is elevated in the brain as the central nervous system (CNS) is still developing and maturing, causing the brain to be more susceptible to insults, including alcohol-induced impairments (Arain M et al., 2013). Studies have shown that binge-like alcohol consumption among adolescents leads to persisting adverse neurological consequences (Crews FT et al., 2016). More specifically, the hippocampal region of the brain is particularly vulnerable to the negative consequences of binge alcohol consumption, impacting learning (Swartzwelder HS et al., 2015), and memory (Macht V et al., 2020;Risher ML et al., 2013). These studies utilize the well-established rodent model of adolescent intermittent ethanol (AIE) exposure to better understand the effects of excessive alcohol use during adolescences on the hippocampal microenvironment.

Dysregulation of pro- and anti-inflammatory cytokines, neurotrophic responses, and neurogenesis in the hippocampus have all been suggested to contribute to hippocampal impairments observed after excessive alcohol use (Crews FT et al., 2019;Geil CR et al., 2014); however, fewer studies have addressed the hypothesis that sex is an important biological variable in the persisting dysregulation of the neurogenic niche after excessive adolescent alcohol consumption (Robinson DL et al., 2021) Therefore, the present study aims to characterize whether there are sex-specific effects on neurogenesis and inflammatory/neurotrophic factor balance after AIE. In the hippocampus of rodents, neurogenesis is restricted to the subgranular zone (SGZ) of the dentate gyrus (DG) (Abrous DN et al., 2005;Macht V et al., 2020). Once the cells are formed they integrate into the granular layer of the DG and extend their axons and dendrites to their targets (von Bohlen Und Halbach O, 2007). There are several stages of neurogenesis in the DG that can be differentiated using a multitude of biological markers (Semerci F and Maletic-Savatic M, 2016). During the proliferation phase, Ki-67 antibody is able to recognize nuclear antigen present in proliferating cells that is not present in resting cells (Gerdes J et al., 1983) and is expressed in abundance, as during this stage neuronal cells are actively dividing. During the differentiation stage the transient amplifying cells differentiate into immature neurons and commit to a neuronal lineage in the SGZ. During this phase, doublecortin (DCX) is expressed transiently by migrating neuroblasts as they move to the granule cell layer of the DG. DCX is associated with morphological changes and migration (LoTurco J, 2004). Additionally, Sox2 is expressed in embryonic early neural precursors of the ventricular zone which is the embryonic layer of tissue that contains the progenitor stem cells of the central nervous system, during neurogenesis cell differentiation (Ferri AL et al., 2004). Sox2, DCX, and Ki-67 are all used in these studies to understand AIE’s impact on adult neurogenesis.

Neurogenesis in the hippocampus of rodents is regulated by neurotrophic factors, which were originally characterized by their ability to aid in neuronal development, survival, and differentiation and regulate brain plasticity (Davies AM et al., 1986;Lin LF et al., 1993). Considering AIE’s prolonged effects on neurogenesis (Macht V,Crews FT and Vetreno RP, 2020), an imbalance in neurotrophic factors and inflammatory cytokines are a potential neural mediator of interest to understanding the persisting effect of alcohol consumption on neurogenesis. Two neurotrophins that have a role in neurogenesis are brain-derived neurotrophic factor (BDNF) (Zagrebelsky M and Korte M, 2014) and glial-derived neurotrophic factor (GDNF) (Barak S et al., 2019;Lin LF,Doherty DH,Lile JD,Bektesh S and Collins F, 1993). Moreover, both GDNF and BDNF have previously been reported as being depressed by chronic long-term binge drinking and conversely have been shown to influence alcohol consumption and other related behaviors (Liran M et al., 2020). In fact, both BNDF and GNDF play important roles in alcohol use disorders and addictive behavior (Barak S,Ahmadiantehrani S,Logrip ML and Ron D, 2019;Heberlein A et al., 2010). Interestingly, there are also sex differences that have been reported on trophic and proinflammatory responses in the CNS (Chan CB and Ye K, 2017;Chistyakov DV et al., 2018;Osborne BF et al., 2018;Rossetti AC et al., 2019;Sardar R et al., 2021).

Historically, studies have shown that males tend to binge drink more than females (Nolen-Hoeksema S, 2004;Wilsnack RW et al., 2009), consequently, female subjects have not always been prioritized in studies of binge-like alcohol consumption, especially in pre-clinical studies. However, a youth risk behavior survey in 2019, showed that females had a significantly higher prevalence for alcohol use reporting at 31.9% than males at 26.4%, and were more likely to engage in binge drinking (14.6% vs. 12.7%) (Jones CM CH, Deputy NP, et al. , 2020). With the rise in binge drinking among female adolescents it has become imperative to study the persisting impacts of alcohol while including sex as a biological variable. Moreover, preclinical studies are increasingly reporting distinct sex differences in both behavioral and neurobiological responses to adolescent alcohol exposure (Robinson DL,Amodeo LR,Chandler LJ,Crews FT,Ehlers CL,Gómez-A A,Healey KL,Kuhn CM,Macht VA,Marshall SA,Swartzwelder HS,Varlinskaya EI and Werner DF, 2021). Those differences include spatial navigational learning deficits mainly driven by females (Macht V,Elchert N and Crews F, 2020), increased exploratory behavior in female rats (Healey KL et al., 2022), decreased dominant behaviors in females (Macht V,Elchert N and Crews F, 2020), reduced anxiety in females compared to males (Healey KL,Kibble SA,Bell A,Kramer G,Maldonado-Devincci A and Swartzwelder HS, 2022), and exacerbated microglial responses in females (Nwachukwu KN et al., 2022). Moreover, clinical findings from the National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA) cohort suggested that the effects of adolescent alcohol consumption caused weaker brain network maturation and functional connectivity in adolescent females than males, and the effects of adolescent alcohol use overall were more pronounced in females than in males (Zhao Q et al., 2021). Taking into consideration these recent findings highlighting sex differences in AUDs and AUD-models, this study seeks to both examine the female response to AIE as well as to compare it directly to the male response.

This current study builds on previous studies that have shown that AIE causes long term effects on neurogenesis in males by assessing these effects in females as well directly comparing the male and female response (Macht V et al., 2021;Reitz NL et al., 2021;Swartzwelder HS et al., 2019). To assess the persisting sex-specific effects of AIE on neurogenesis, pro-inflammatory and anti-inflammatory cytokines, and neurotrophic factors, we utilized the rat AIE oral gavage model that recapitulates the intermittent drinking habits of human adolescents (Healey KL et al., 2020;Healey KL,Kibble SA,Bell A,Kramer G,Maldonado-Devincci A and Swartzwelder HS, 2022;Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022;Swartzwelder HS,Healey KL,Liu W,Dubester K,Miller KM and Crews FT, 2019). This current study is an expansion of our recent work that indicated male and female rats have similar increased neuroimmune responses in adulthood following AIE, however some glial responses were more robust or only found in females (Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022). Here we test the hypothesis that sex is a biological factor that modifies AIE-induced impairment of neurogenesis and dysregulation of pro- and anti-inflammatory cytokines and neurotrophic factors.

Experimental Procedures

Adolescent Intermittent Ethanol (AIE)

All the techniques and methods used in these experiments were approved by the Duke University Institutional Animal Care and Use Committee (Protocol Registry Number A159-18-07) and were conducted in accordance with the guidelines of the American Association for the Accreditation of Laboratory Animal Care and the National Research Council’s Guide for Care and Use of Laboratory Animals (NRC, 2011). Adolescent male (n=32) and female (n=32) Sprague-Dawley rats (Charles River, Raleigh, NC, USA; postnatal day (PND) 16) were allotted 7 days to habituate before weening and an additional 7 days to acclimate to the vivarium on a reversed 12-hour light:dark cycle before dosing. In each cage, two rats were housed with ad libitum access to food and water. Rats were pair housed based on sex as well as treatment groups. Starting on PND30, ethanol (Decon Labs, Prussia, PA, US) 5.0 g/kg (35% ethanol v/v in water at 18.12 ml/kg) or isovolumetric water 18.12 ml/kg was administered via intragastric gavage during adolescence (PND30-46) as previously described for the AIE model (Healey KL et al., 2021;Swartzwelder HS,Healey KL,Liu W,Dubester K,Miller KM and Crews FT, 2019). All animals in the study survive the procedure as the dose is well below the LD50 for rats (Wiberg GS et al., 1970). This dose has been chosen to mimic high BECs that are often achieved in juveniles that binge drink (Ruetsch V et al., 2021;Vinader-Caerols C et al., 2017). For Experiment 1 (Immunohistochemistry (IHC) experiments), rats were administered a total of 10 doses on a 2-days on, 1-day off, 2-days on, 2-days off schedule for a duration of 16 days until all doses were administered. Rats were weighed prior to every dose to receive the correct dosage. Following dosing, animals matured into adulthood and at PND 77 were euthanized by paraformaldehyde perfusion for the IHC experiments. For Experiment 2 (ELISA experiments) rats were also given the same habituation timeline as the rats in Experiment 1 prior to dosing and the same dosage of ethanol and water, however, rats were administered a total of 14 doses on a 2-days on, 1-day off, 2-days on, 2-days off schedule for a duration of 23 days. Subsequently, animals matured into adulthood and at PND 84 were euthanized by rapid decapitation for ELISA experiments. Adulthood in rats is considered at approximately PND 65 (McCutcheon JE and Marinelli M, 2009). The dosing schedule for Experiment 2 differ from Experiment 1 because of the new standards that were recently established by the Neurobiology of Adolescent Drinking in Adulthood (NADIA) consortium to decrease the stress of animal delivery and to enhance the AIE procedure timeline to include early and late-stage adolescence. We do not expect the additional AIE doses to effect outcome as the NADIA has found several different AIE exposure periods to produce robust effects (Crews FT,Robinson DL,Chandler LJ,Ehlers CL,Mulholland PJ,Pandey SC,Rodd ZA,Spear LP,Swartzwelder HS and Vetreno RP, 2019).

After the paraformaldehyde perfusions of the Experiment 1 group, brains were dissected out, postfixed in 4% (w/v) paraformaldehyde for 24 hours, and placed in cryopreserve (1mM polyvinyl-pyrrolidone, 50% (v/v) ethylene glycol in 0.2M phosphate buffer, pH=7.4) until further processing. For Experiment 2, following rapid decapitation, the hippocampus from both hemispheres were microdissected out and snap frozen until homogenized. Two independent groups of animals from the Experiment 1 and Experiment 2 cohorts were utilized to collect blood samples for blood ethanol concentrations (BECs) to diminish stress and anxiety amongst the experimental animals. The average BECs were similar for both sexes, as the mean BEC achieved for male sentinel rats was 220.8 mg/dL and 210.6 mg/dL for female rats, 60 minutes following AIE exposure, congruent with our previous studies (Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022). An unpaired t-test did not reveal any significant difference between the BEC levels that were achieved between cohorts (data not shown). Although these animals were run separately, it is consistent with our recent studies that also indicate similar metabolism across sexes (Healey KL,Kibble S,Bell A,Hodges S and Swartzwelder HS, 2021;Healey KL et al., 2020). A random number generator was used to assign experimental cohorts with cage numbers to blind experimenters to the treatment groups. All measurements for immunohistochemistry and ELISA studies were completed in adulthood succeeding AIE exposure to determine the persisting effect of adolescent exposure following a period of discontinuation from ethanol discontinuation.

Immunohistochemistry & Quantification

The methods utilized for the immunohistochemistry protocols were similar to previous studies (Grifasi IR et al., 2019;Marshall SA et al., 2016;Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022;Swartzwelder HS,Healey KL,Liu W,Dubester K,Miller KM and Crews FT, 2019). Briefly, the whole brain was sectioned at a series of 1:12 at 40μm thickness with a compresstome VF-300 microtome (Precisionary Instruments Inc., Greenville, NC) (Grifasi IR,McIntosh SE,Thomas RD,Lysle DT,Thiele TE and Marshall SA, 2019;Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022). The sections were stored in cryopreserve at −20 °C until further processing. After a series of phosphate buffered saline (PBS) washes, using 0.6% H₂O₂ to quench endogenous peroxidases, and blocking non-specific antibody binding (0.1% triton X and 3% goat serum in 0.1M PBS), tissue series were incubated in 1:400 rabbit Doublecortin (DCX; abcam, Waltham, MA, ab18723), 1:200 rabbit SRY-box 2 (SOX2; MilliporeSigma, Burlington, MA, AB5603) or 1:200 rabbit nuclear protein Ki-67 (Ki-67; ThermoFisher Scientific, Waltham, MA, MA5-14520) at 4 °C for 48hrs. The primary antibodies, DCX, SOX2 and Ki-67, were chosen from commercially available sources specific for neurogenesis properties, utilizing colabeling and western blot techniques for validation. Following incubation in the primary antibody, sections were then washed in another series of PBS and then incubated in 1:2000 goat anti-rabbit secondary antibody (Vector Laboratory, Burlingame, CA) for an hour. The sections were put through another series of PBS washes before using an ABC peroxidase staining kit (Thermo Scientific, Rockford, IL; 1 hour) to form a horseradish peroxidase complex with the chromogen 3,3’-Diaminobenzidine (DAB;ACROS organics, Morris Plain, NJ). Sections were then washed in PBS, mounted, and coverslipped with Cytoseal (Thermo Scientific).

Photomicrographs of the DG subregion of the hippocampus were captured using a 10X and 40x objective on a B120 microscope (AmScope, Irvine, CA) with an attached MU500 AmScope digital camera as previously described (Grifasi IR,McIntosh SE,Thomas RD,Lysle DT,Thiele TE and Marshall SA, 2019;Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022). The experimenters were blinded to the various treatment groups during quantification. The open-source software Qupath 0.2.3 was utilized to assess cell counts SOX2+ and immunoreactivity in DCX+ and SOX2+ cells. (Bankhead P et al., 2017). The DG of dorsal hippocampal subregion was individually traced on each photomicrograph between Bregma −2.30mm and −4.52mm (Paxinos G, & Watson, C. , 2009). The density of DAB+ pixels was used as a proxy of DCX and SOX2 immunoreactivity and was quantified in each region of interest by a user defined threshold (% area). SOX2+ cell counts were automated because it is a marker of early stem cells and morphology, and density is not a factor in getting precise counts using QuPath software. This output combines the user-defined threshold for DAB using intensity and color threshold for saturation and hue with a window for the size of the cell based on the area of positive pixels. However, DCX+ cells were manually counted due to morphology and density of the cell population. Likewise, due to the clustering of Ki-67+ cells via proliferation, positive cells were manually counted to decrease discrepancies. All cell counts were expressed as DCX+, SOX2+ or Ki-67+ cells/section. Animals with less than 7 usable hippocampal sections were excluded from the analysis.

Enzyme Linked Immunosorbent Assay (ELISA)

The hippocampus from the right and left hemisphere of the rats were processed for ELISA similar to the protocols reported previously (Marshall SA et al., 2013;Marshall SA et al., 2020). Hippocampal tissue was homogenized in lysis buffer that was ice cold (1 mL of buffer/50mg of tissue; pH = 7.40– 25mM HEPES (MilliporeSigma), 0.1% CHAPS (Alfa Aesar; Haverhill, MA), 5mM MgCl₂·6H₂0 (MilliporeSigma), 1.3mM EDTA (Avantor; Radnor Township, PA), 1mM EGTA (MilliporeSigma), 10 μg/mL Pepstatin A (Thermo Scientific), 10 μg/mL Aprotinin (Thermo Scientific), 10 μg/mL Leupeptin (Thermo Scientific), 1mM PMSF (MilliporeSigma)), then the supernatant was transferred to a new microcentrifuge tube and stored at −80. Rat tumor necrosis factor-α (TNF-α; Invitrogen, #ERA57RB; Waltham, MA, USA), rat interleukin-1β (IL-1β; Invitrogen, #ERIL1B), rat interleukin-10 (IL-10; Invitrogen, #ERA24RB), and rat Transforming Growth Factor-β1 (TGF-β1; Invitrogen, #ERA56RB) protein concentrations were determined via ELISA according to the manufacturer’s instructions. These cytokines were chosen to assess pro-inflammatory and anti-inflammatory responses in adulthood following AIE respectively. Additionally, rat Brain Derived Neurotrophic Factor (BDNF; BOSTER, #EK0308; Pleasanton, CA), and rat Glial Cell-Derived Neurotrophic Factor (GDNF; BOSTER, #EK0363) were measured in the hippocampus. All samples and standards for each ELISA kit were run in duplicate. Absorbance was measured at 450 nm on a BioTek Synergy HT (Agilent; Santa Clara, CA) microplate reader. Cytokine concentrations were normalized to the total protein content as determined by a Pierce BCA Protein Assay Kit (Thermo Scientific) and reported as pg/mg of total protein.

Statistical Analysis

All data were graphed and analyzed using GraphPad Prism 9.1.2 (GraphPad Software, Inc. La Jolla, Ca). Data were analyzed using two-way analyses (treatment × sex) of variance (ANOVAs) followed by Tukey test post hoc analyses if there was a significant interaction. All our samples were tested for normal distribution and were all satisfied using the Kolmogorov-Smirnov and Shapiro-Wilk normality tests. Boxes represent the median and the 25th and 75th quartiles and the whisker showing the 5th and 95th percentile in the graphs. Effects were considered significant if p < 0.05.

Results

AIE causes a persistent reduction of DCX+ immunoreactivity and DCX+ cells in the subgranular zone of the dentate gyrus in male and female rats in adulthood

The sex-specific effects of AIE on DCX immunoreactivity were assessed utilizing densitometry as a correlate of neurogenesis (Figure 1). Two-way ANOVA (treatment × sex) showed a main effect of AIE in the DG subregion [F_(1,22) =35.22, p < 0.0001]; however, there was no interaction [F_(1,22) = 0.02516, p = 0.8754] or main effect of sex [F_(1,22) = 4.673, p = 0.0418 (Figure 1E). Thus, AIE led to a 35% reduction in DCX+ immunoreactivity in the SGZ of the DG in both males and females. Because changes in neuronal morphology and neuronal numbers can influence DCX immunoreactivity, manual cell counts were conducted to determine whether changes in immunoreactivity were associated with newly generated neurons. Based on the cell counts with in the DG subregion, a two-way ANOVA (treatment × sex) showed a main effect of AIE [F_(1,22) = 22.10, p = 0.0001]; however, there was no interaction [F_(1,22) = 0.1659, p = 0.6877] or main effect of sex [F_(1,22) = 0.03995, p = 0.8434] on the number of DCX+ cells (Figure 1F). AIE also led to a 10% decrease in the number of DCX+ cells in the SGZ of the DG in both males and females.

Figure 1.

Schematic of experimental timeline and treatment for experiment 1 and experiment 2. (A) Experiment 1 immunohistochemistry (B) Experiment 2 ELISA.

AIE causes a persistent decrease in SOX2+ cells and immunoreactivity in the dentate gyrus of male and female rats in adulthood

The sex-specific effects of AIE on SOX2 immunoreactivity were assessed utilizing densitometry as a marker for early neurogenesis via neural stem cells (Figure 2). A two-way ANOVA (treatment × sex) showed a main effect of AIE in the DG subregion [F_(1,23) = 33.69, p < 0.0001]; however, there was no interaction [F_(1,23) = 0.9233, p = 0.3466] or main effect of sex [F_(1,23) = 3.590, p = 0.0708 (Figure 2E). Thus, similar to its effect on DCX+ immunoreactivity, AIE also led to a 2-fold decrease in SOX2+ immunoreactivity in the DG of both males and females.

Figure 2.

Adolescent intermittent ethanol (AIE) exposure causes a persistent reduction of DCX+ cells and immunoreactivity in the adult dentate gyrus of male and female rats. Photomicrographs indicate doublecortin positive (DCX+) cells are decreased in the subgranular zone of the dentate gyrus (DG) hippocampal subregion of adolescent intermittent ethanol (AIE) exposed male and female rats (B,D) in adulthood in comparison to their adolescent intermittent water (AIW) exposed controls (A,C) respectively. A significant reduction was observed in (E) DCX+ immunoreactivity and (F) DCX+ cell counts in the subgranular zone of both sexes. Images were taken at 10x and 40x magnification. Granular cell layer, scale bar = 40μm; inset scale bar = 10μm; Boxes represent the median and the 25th and 75th quartiles and the whisker showing the 5th and 95th percentile. *p < 0.05 AIW vs. AIE; (white bars = AIW; grey bars = AIE; black dots = animal averages)

Automated cell counts were conducted to determine whether changes in immunoreactivity were associated with neural stem cell differentiation. In the DG subregion, a two-way ANOVA (treatment × sex) showed a main effect of AIE [F_(1,23) = 11.50, p = 0.0025]; however, there was no interaction [F_(1,23) = 0.7893, p = 0.3835] or main effect of sex [F_(1,23) = 1.671, p = 0.2089] on the number of SOX2+ cells (Figure 2F). Thus, AIE led to a 10–15% decrease in SOX2+ immunoreactivity in the DG of both male and female rats.

AIE decreases Ki-67+ cells in the dentate gyrus of male and female rats

The sex-specific effects of AIE on KI-67+ cells were assessed utilizing manual cell counts to determine neurogenesis proliferation (Figure 3). A two-way ANOVA (treatment × sex) showed a main effect of AIE in the DG subregion [F_(1,22) = 29.83, p < 0.0001] and main effect of sex [F_(1,22) = 11.09, p = 0.0030]; however there was no interaction [F_(1,22) = 2.525, p = 0.1263 (Figure 3E). Thus, AIE led to a significant 20% reduction of Ki-67+ cells in the SGZ of the DG in both males and females. The main effect of sex revealed a significant difference between AIE males compared to their AIW controls p < 0.0005 and AIE females in compared to their AIW controls p < 0.0409.

Figure 3.

AIE decreases SOX2+ cells and immunoreactivity in the DG of male and female rats following abstinence. Photomicrographs suggest SRY-Box Transcription Factor 2 positive (SOX2+) cells are reduced in the DG hippocampal subregion of AIE exposed male and female rats (B,D) in adulthood in comparison to their AIW exposed controls (A,C) respectively. A significant reduction was observed in (E) SOX2+ immunoreactivity and (F) SOX2+ cell counts in the DG subregion of both sexes ± SEM. Images were taken at 10x magnification. Scale bar = 40μm; *p < 0.05 AIW vs. AIE; Boxes represent the median and the 25th and 75th quartiles and the whisker showing the 5th and 95th percentile. (white bars = AIW; grey bars = AIE; black dots = animal averages)

Anti-inflammatory cytokines are not altered in the hippocampus of male or female rats following AIE maturation

Analysis of the sex-specific effects of AIE on anti-inflammatory cytokines protein expression were assessed utilizing ELISA assays (Figure 4). In the IL-10 ELISA assay a two-way ANOVA (treatment × sex) indicated an interaction [F_(1,31) = 5.931, p = 0.0208]; however, there was no main effect of AIE [F_(1,31) = 0.08028, p = 0.7788] or main effect of sex [F_(1,31) = 0.08735, p = 0.7695 (Figure 4A). However, a Tukey post hoc analysis revealed no distinguishable effects. Additionally, in the TGF-β1 ELISA assay a two-way ANOVA (treatment × sex) revealed no interaction in the hippocampus [F_(1,31) = 0.2381, p = 0.6290], nor main effect of AIE [F_(1,31) = 0.02696, p = 0.8706] or sex effect [F_(1,31) = 0.01034, p = 0.9197 (Figure 4B).

Figure 4.

AIE decreases Ki-67+ cells in the adult DG of male and female rats. Photomicrographs suggest nuclear protein Ki-67 positive (Ki67+) cells are reduced in the subgranular zone of the DG hippocampal subregion of AIE exposed male and female rats (B,D) in adulthood in comparison to their AIW exposed controls (A,C) respectively. A significant reduction was observed in (E) Ki-67+ cell counts in the subgranular zone in both sexes. Images were taken at 10x magnification. Scale bar = 40μm; *p < 0.05 AIW vs. AIE; #p < 0.05 males vs. females; Boxes represent the median and the 25th and 75th quartiles and the whisker showing the 5th and 95th percentile. (white bars = AIW; grey bars = AIE; black dots = animal averages)

Pro-inflammatory cytokines are increased in the hippocampus of only male rats following AIE maturation

Furthermore, analysis of the sex-specific effects of AIE on pro-inflammatory cytokines protein expression were also assessed utilizing ELISA assays. A two-way ANOVA (treatment × sex) of TNF-α ELISA indicated an interaction in the hippocampus [F_(1,29) = 0.0034 p = 0.0034]; however, there was no main effect of AIE [F_(1,29) = 0.8416, p = 0.3665] or main effect of sex [F_(1,29) = 0.09022, p = 0.7660 (Figure 4C). A Tukey post hoc analysis revealed that there was a significant increase in TNF-α cytokine protein in male rats, but not female rats exposed to AIE, compared to their AIW controls. Lastly, in the IL-1β ELISA assay a two-way ANOVA (treatment × sex) indicated an interaction in the hippocampus [F_(1,30) = 5.221, p = 0.0296] and a main effect of AIE [F_(1,30) = 4.874, p = 0.0351]; however there was no main effect of sex [F_(1,30) = 1.198, p = 0.2825 (Figure 4D). A Tukey post hoc analysis revealed that there was a significant increase in IL-1β cytokine protein in male rats, but not female rats exposed to AIE, compared to their AIW controls.

Neurotrophic factors are not altered in the hippocampus of male or female rats following AIE maturation

The neurotrophic factors BDNF and GDNF were assessed using ELISA to determine whether sex-specific effects of AIE are a factor in the neurotrophic responses (Figure 4). A two-way ANOVA (treatment × sex) of the BDNF ELISA showed no interaction in the hippocampus [F_(1,31) = 0.2776, p = 0.6020], no main effect of AIE [F_(1,31) = 1.145, p = 0.2929] or main effect of sex [F_(1,31) = 2.694, p = 0.1108] (Figure 4E). A two-way ANOVA (treatment × sex) of the GNDF ELISA showed no interaction in the hippocampus [F_(1,31) = 1.394, p = 0.2468], no main effect of AIE [F_(1,31) = 1.262, p = 0.2699] or main effect of sex [F_(1,31) = 1.520, p = 0.2268] (Figure 4F). AIE did not alter BNDF or GDNF protein concentrations in the hippocampus of either male or female rats.

Discussion

It was once thought that neurons within the brain were unable to regenerate. However, studies over the years have characterized the DG subgranular zone of the hippocampus as a site for adult neurogenesis, differentiated from progenitor stem cells (Eriksson PS, 2003). Heavy adolescent alcohol exposure has been shown to perturb the division and migration of hippocampal preneuronal progenitor cells, ultimately interfering with neurogenesis seen in both nonhuman primates (Taffe MA et al., 2010) and rodents (McClain JA et al., 2011). Although those previously mentioned studies were carried out in acute alcohol exposure models, most of the previous studies of AIE effects have excluded female subjects. However, with the increase in female adolescent binge drinking trends, and the obvious general scientific need to understand sex as a biological variable, females have begun to be included in AIE pre-clinical studies as it relates to behavior (Healey KL,Kibble SA,Bell A,Kramer G,Maldonado-Devincci A and Swartzwelder HS, 2022;Macht V,Elchert N and Crews F, 2020;Reitz NL,Nunes PT and Savage LM, 2021) and neuroimmune responses (Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022). In addition, some studies that have shown AIE effects in males but not females. For example, Vore and colleagues observed an increase in blood brain barrier permeability in the hippocampus and ventral tegmental area of AIE male rats due to AIE induced VEGFa and PDGFRβ protein alterations (Vore AS et al., 2022). The present study aimed to determine the effects of AIE on hippocampal neurogenesis, cytokine inflammatory responses, and the neurotrophic factor niche, while considering sex-specific effects. The major findings from this study are 1) male and female rats have persisting effects on neurogenesis as indicated by a significant decrease in DCX+, SOX2+, and Ki-67+ cells in the dentate gyrus subregion of the hippocampus, 2) AIE had no persisting impact on anti-inflammatory cytokines IL-10 and TGF-β1 protein concentrations in the hippocampus of male or female rats, 3) Pro-inflammatory cytokines TNF-α and IL-1β protein concentrations were increased in the hippocampus of AIE male rats but not in AIE female rats compared to their respective AIW controls 4) Neurotrophic factors BDNF & GDNF protein concentrations were not impacted in adulthood following AIE in either sex.

Our AIE neurogenesis data align with previous studies that have observed the impact of AIE on neurogenesis in adolescent male rats, which have consistently demonstrated a reduction in DCX+, SOX2+, and Ki-67+ neurogenic markers in the DG of the hippocampus of male rats (Crews FT et al., 2006;Hansson AC et al., 2010;Kempermann G et al., 2018;Macht V,Crews FT and Vetreno RP, 2020;Swartzwelder HS,Healey KL,Liu W,Dubester K,Miller KM and Crews FT, 2019). Additionally, other ethanol administration models such as intraperitoneal administration using the persisting AIE model in male rats demonstrated similar decreases of Ki-67+ and DCX+ cells in the dentate gyrus (Sakharkar AJ et al., 2016), as well, acute and chronic oral gavage of male rats (Nixon K and Crews FT, 2002) yielding similar reductions in neurogenesis using the bromodeoxyuridine-positive (BrdU+) neurogenic marker. However, to our knowledge our current findings are the first to report the persisting impact of AIE on neurogenic markers in the DG hippocampal subregion of adult female rats. A previous report with all male rats indicated that after AIE reduced DCX+, Ki-67+ and SOX2+ expression in the subventricular zone and in the DG of male rats (Liu W and Crews FT, 2017). These findings were also consistent with our previous study which showed a similar reduction in DCX+ cells in the DG in male adult rats (Swartzwelder HS,Healey KL,Liu W,Dubester K,Miller KM and Crews FT, 2019). The present study adds to this body of work by indicating that female rat neurogenic responses are similar to male rats as indicated by significant reductions of DCX+, SOX2+, and Ki-67+ cells in the DG. Reductions in SOX2+ cells and immunoreactivity can impair the ability of neural stem cells and progenitor cells to self-renew and proliferate, leading to dysregulation of neurogenesis (Ferri AL,Cavallaro M,Braida D,Di Cristofano A,Canta A,Vezzani A,Ottolenghi S,Pandolfi PP,Sala M,DeBiasi S and Nicolis SK, 2004). Likewise, a decrease in Ki-67+ cells is associated with perturbed proliferation of new progenitor cells representing a decrease in neurogenesis (Reif A et al., 2006). Additionally, a reduction in DCX+ has been shown to be an indicator of the reduction of neurogenesis in the dentate gyrus (Brown JP et al., 2003). Taken together the observed reduction of all three markers suggests an inability of cells to regenerate and proliferate following AIE exposure.

Neurogenesis can be modulated by inflammatory cytokines in response to insult or injury, altering neuronal proliferation and differentiation (Borsini A et al., 2015). More specifically, alcohol-related experiments have shown an increase in hippocampal anti-inflammatory cytokine IL-10 expression one hour after an intoxicating dose of ethanol (Neupane SP et al., 2016) and 7-days following a 4-day binge exposure indicated a significant increase in IL-10 and TGF-β1 anti-inflammatory hippocampal cytokines (Marshall SA,McClain JA,Kelso ML,Hopkins DM,Pauly JR and Nixon K, 2013). However, in this study, we report no persisting changes in IL-10 and TGF-β1 protein expression levels in the hippocampus of male or female rats in adulthood following AIE. Additionally, when microglia are primed, they can release pro-inflammatory cytokines such as TNF-α and IL-1β (Glass CK et al., 2010), and a 10-day daily ethanol exposure in male mice produced a significant increase in TNF-α but not IL-1β protein expression in the brain (Qin L et al., 2008). The present findings describe significant increases in TNF-α and IL-1β in the hippocampus of male but not female rats in adulthood after AIE. Additionally, in our previous study we reported an increase of Iba-1 in the hippocampus of female rats but not in males (Nwachukwu KN,King DM,Healey KL,Swartzwelder HS and Marshall SA, 2022). Although that finding may appear inconsistent with the present observation of elevated TNF-α and IL-1β protein expression levels females, it is important to note that the microglia changes were regionally specific in our previous study, whereas in this study the whole hippocampus was analyzed. In any case, the present findings suggest an underlying association between TNF-α and IL-1β protein expression and neurogenesis dysregulation mainly driven in male rats that may not be microglia dependent. The upregulation of TNF-α and IL-1β protein expression could contribute to neuronal damage mechanisms hindering neurogenesis further following AIE.

Neurotrophic factors support neuronal development, survival and differentiation (Lin LF,Doherty DH,Lile JD,Bektesh S and Collins F, 1993) and the ability to regulate neuronal plasticity (Zagrebelsky M and Korte M, 2014). More specifically, BDNF regulates both excitatory and inhibitory synaptic transmission in the adult brain (Wardle RA and Poo MM, 2003), and the highest expression levels of BDNF are found in hippocampal neurons (Timmusk T et al., 1993). Previous studies have reported that epigenetic modifications succeeding AIE lead to a reduction in BDNF expression in the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions of the hippocampus utilizing BDNF mRNA (Sakharkar AJ,Vetreno RP,Zhang H,Kokare DM,Crews FT and Pandey SC, 2016). The same study also reported inhibition of neurogenesis in the hippocampus via DCX and Ki-67 immunohistochemistry in adulthood (Sakharkar AJ,Vetreno RP,Zhang H,Kokare DM,Crews FT and Pandey SC, 2016), aligning with the present findings. Moreover, Scheidt et al. observed a correlation between AIE and lower levels of BDNF in the hippocampus of male adult rats with 1.0g/kg but not 3.0g/kg (Scheidt L et al., 2015). The current data aligns with the BDNF response to higher doses of ethanol reporting no significant reduction in BDNF protein expression in the adult hippocampus of male rats, but this report indicates the lack of persisting effects is also true for female rats. The inconsistency between our work and those that suggest a decrease in BDNF could be due to the differences in methodological approaches. Our studies and Scheidt examined BDNF protein in homogenized samples of the dorsal hippocampus while and others look at mRNA or used immunohistochemistry to detect BDNF in the hippocampus (Sakharkar AJ,Vetreno RP,Zhang H,Kokare DM,Crews FT and Pandey SC, 2016;Sampedro-Piquero P et al., 2022). Moreover, the reported increases in BDNF after adolescent alcohol exposure were regionally specific and may have been washed out in the homogenization process (Sakharkar AJ,Vetreno RP,Zhang H,Kokare DM,Crews FT and Pandey SC, 2016;Sampedro-Piquero P,Moreno-Fernández RD,Begega A,López M and Santín LJ, 2022). Likewise, this study indicated no enduring effects of AIE on GDNF. Studies in adult rats have shown that GDNF expression in the ventral tegmental area is upregulated after two-bottle choice intermittent alcohol consumption one week after the last exposure but not after seven weeks (Ahmadiantehrani S et al., 2014). The difference in these findings suggest that timing, regional specificity, and the age of the animal are variables that may account for the variations in GDNF expression. Finally, human clinical studies reported no significant reduction in GDNF or BDNF expression in the serum of chronic alcohol users during withdrawal (Heberlein A,Muschler M,Wilhelm J,Frieling H,Lenz B,Groschl M,Kornhuber J,Bleich S and Hillemacher T, 2010). Considering the contradictory findings within alcohol-induced changes in growth factors like GDNF and BDNF (Liran M,Rahamim N,Ron D and Barak S, 2020), future studies should focus on how ethanol doses and age impact transcription versus translation following AIE.

The present findings add to the current body of work by identifying the impact of AIE on neurogenesis and the cytokine and neurotrophic niche while considering sex as a biological variable. These findings will aid in the development of hypotheses and procedures for future studies of sex differences in alcohol responsiveness, particularly as relates to neurogenic and inflammatory mechanisms. Altogether, our findings suggest that AIE does not have a long-term impact on neurotrophic levels but still causes a significant reduction in neurogenesis. The neurogenic impairment was not sex specific, but the increase in pro-inflammatory cytokines observed in males may contribute to the male neurogenic deficits. More studies will need to be done to focus on the underlying mechanisms of the neurogenic impairment. A long-term impairment in neurogenesis may influence both behavioral maladaptations and neurodegeneration in adulthood.

Figure 5.

Anti-inflammatory cytokines and neurotrophic factors were not altered in the hippocampus of male or female rats, but pro-inflammatory cytokines were increased in the hippocampus of male rats following AIE exposure. We observed no changes in ELISA protein concentrations on anti-inflammatory cytokines (A) rat Interleukin-10 (IL-10) or (B) rat Transforming Growth Factor-β1 (TGF-β1) in the hippocampus of male or female rats following AIE abstinence. However, we did observe a significant increase in rat Tumor Necrosis Factor-α (TNF-α) (C) and Interleukin-1β (IL-1β) in the hippocampus of male AIE rats but not female AIE rats (D). Additionally, there were no persisting impacts of AIE on neurotrophic factors (E) rat Brain Derived Neurotrophic Factor (BDNF) nor (F) rat Glial Cell-Derived Neurotrophic Factor (GDNF) in the hippocampus of male or female rats following AIE abstinence. #p < 0.05 males vs. females; Boxes represent the median and the 25th and 7th quartiles and the whisker showing the 5th and 9th percentile. (white bars = AIW; grey bars = AIE; black dots = animal averages)

Highlights:

• Neurogenesis was reduced in the hippocampus of female and male rats following AIE.

• AIE increased pro-inflammatory cytokines in male rats but not female rats.

• AIE did not alter anti-inflammatory cytokine protein expression in either sex.

• AIE did not alter neurotrophic factor protein expression in either sex.

Abbreviations

AIE

Adolescent Intermittent Ethanol

AIW

Adolescent Intermittent Water

ANOVA

Analysis of Variance

AUD

Alcohol Use Disorder

BDNF

Brain-Derived Neurotrophic Factor

BEC

Blood Ethanol Concentrations

BrdU

Bromodeoxyuridine

CA1

Cornu ammonis 1

CA3

Cornu ammonis 3

CDC

Centers for Disease Control and Prevention

CNS

Central Nervous System

DAB

3,3’-Diaminobenzidine

DCX

Doublecortin

DG

Dentate Gyrus

ELISA

Enzyme Linked Immunosorbent Assay

GDNF

Glial-Derived Neurotrophic Factor

IHC

Immunohistochemistry

IL-1β

Interleukin-1β

IL-10

Interleukin-10

NADIA

Neurobiology of Adolescent Drinking in Adulthood

NCANDA

National Consortium on Alcohol and Neurodevelopment in Adolescence

PBS

Phosphate Buffered Saline

PND

Postnatal Day

SGZ

Subgranular Zone

SOX2

SRY-box 2

TGF-β1

Transforming Growth Factor-β1

TNF-α

Rat Tumor Necrosis Factor-α