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. 2025 Feb 18;35(2):e70001. doi: 10.1002/hipo.70001

Donepezil Reverses Alcohol‐Induced Changes in Hippocampal Neurogenic and Glial Responses Following Adolescent Intermittent Ethanol Exposure Into Adulthood in Female Rats

Kala N Nwachukwu 1,2, James C Nelson 1, Kennedy M Hill 1, Kennedy A Clark 1, Kati Healey 3, H Scott Swartzwelder 4, S Alex Marshall 1,5,✉
PMCID: PMC11836526  PMID: 39967057

ABSTRACT

Adolescent intermittent ethanol (AIE) exposure leads to persisting increases in glial markers and significantly decreases the neurogenic niche in the dentate gyrus of the hippocampus. Our previous study indicated that donepezil (DZ), a cholinesterase inhibitor, can reverse the AIE effect of decreased doublecortin (DCX), a neurogenic marker, and increased cleaved caspase 3, a marker of apoptosis, in the dentate gyrus of male rats. However, to date, no studies have assessed the effects of DZ on AIE effects in females. The purpose of this study was to determine whether DZ can reverse neuroimmune, neurogenic, and neuronal death effects in adulthood after AIE in female rats. Adolescent female rats were given 14 doses of ethanol (5 g/kg) over 24 days by intragastric gavage. Seventeen days later, DZ (2.5 mg/kg, 1.88 mL/kg, i.g., in water) was then administered daily for 4 days prior to sacrifice. Immunohistochemical techniques were utilized to determine the effects of DZ on AIE‐induced changes in neurogenesis, cell death, glial, and neuroimmune markers. As expected, AIE decreased the neurogenic markers DCX, SOX2, and Ki‐67 in the dentate gyrus and also caused an increase in the glial markers GFAP and Iba‐1 in the hippocampus. The effects of AIE on neurogenic and glial markers were reversed by DZ treatment, but the reversal of AIE effects on glial markers was regionally specific within the hippocampus. Overall, these findings indicate that systemic DZ in adult female rats ameliorates the effects of AIE on neurogenesis, neuronal cell death, neuroimmune markers, and glial activation markers. Future studies will determine if DZ alters hippocampally driven behaviors, as well as the mechanisms underlying donepezil's effects.

Keywords: adolescent, alcohol, astrocytes, hippocampus, microglia, neurogenesis, neuroimmune

Abbreviations

AIE

Adolescent intermittent ethanol

AIF1

Allograft inflammatory factor 1

AIW

Adolescent intermittent water

ANOVA

Analysis of variance

BEC

Blood ethanol concentrations

CA1

Cornu ammonis 1

CA2/3

Cornu ammonis 2/3

CNS

Central nervous system

DAB

3,3′‐Diaminobenzidine

DCX

Doublecortin

DG

Dentate gyrus

DZ

Donepezil

EtOH

Ethanol

GFAP

Glial fibrillary acidic protein

Iba‐1

Ionized calcium binding adaptor molecule 1

IHC

Immunohistochemistry

PBS

Phosphate buffered saline

PND

Postnatal day

SGZ

Subgranular zone

SOX2

SRY‐box 2

VEH

Vehicle

1. Introduction

Binge alcohol consumption continues to be a major problem within the United States, particularly among adolescents and young adults. Alcohol is one of the most widely abused drugs among adolescents due to its ease of accessibility (Warren et al. 2015). According to the 2021 National Survey on Drug Use and Health, among individuals 12 to 20 years of age, 5.9 million reported alcohol use, with 3.2 million reporting binge‐like consumption (SAMHSA 2022). Puberty onset is often considered the start of adolescence (Sawyer et al. 2018), and during adolescence, the brain is highly plastic, hormone levels fluctuate, and there is an elevation of risk‐taking behaviors (Griffin 2017; Laube et al. 2020; Steinberg 2008). One set of adolescent‐typical risky behaviors involves experimenting with alcohol, and in particular with bouts of binge drinking (Richter et al. 2016). Recently, female adolescents have begun to report binge drinking equal to, if not greater than, their male counterparts (Jones et al. 2020), but studies on the effects of alcohol on the brain in preclinical models have predominantly focused on male subjects.

Due to the high vulnerability of the adolescent brain, excessive alcohol consumption during that period has the potential to have persisting effects into adulthood that impact hippocampal integrity (Broadwater et al. 2014; Nwachukwu et al. 2022a) and hippocampally mediated behaviors in rodents (Deschamps et al. 2022; Guo et al. 2022). These studies are consistent with clinical findings that show deficits in short‐ and long‐term memory (Carbia et al. 2017; Mahedy et al. 2018; Mota et al. 2013) as well as decreases in hippocampal volume after adolescent alcohol use (De Bellis et al. 2000; Nagel et al. 2005). One mechanism that has been proposed to underlie hippocampal neurodegeneration in Alcohol use Disorders (AUDs) is the interplay between alcohol‐induced neuroinflammatory pathways and a depression in neurogenesis caused by excessive alcohol consumption (Geil et al. 2014). The current study seeks to determine if those maladaptations are permanent or if they can be reversed with pharmacologic interventions.

Glial cells (microglia, astrocytes, and oligodendrocytes) play a crucial role in the regulation of the neuroimmune response and also have critical homeostatic functions in the central nervous system. Adolescent intermittent ethanol (AIE) exposure, a model of chronic, yet sporadic binge‐like consumption patterns of human adolescents, has been an effective approach to studying the influence of adolescent binge drinking on glia in adulthood (Crews et al. 2019). For example, AIE causes persisting astrogliosis in the hippocampus of female and male rats denoted by an increase in astrocyte density (Nwachukwu et al. 2022b). Likewise, microglia appear to have a long‐lasting proinflammatory, primed response to adolescent alcohol exposure in males (McClain et al. 2011; Vetreno et al. 2017), which was recently shown to be slightly more pronounced in females (Nwachukwu et al. 2022b). Because pharmacologic manipulation of the neuroimmune system can have sex‐dependent effects on alcohol‐related behaviors and maladaptations (Lovelock et al. 2022; Macht et al. 2023), it is important to determine whether neuroimmune modulators can alter AIE effects in adulthood in female animals as well as males.

Neuroimmune dysregulation could also play a role in altering the effects of AIE on the hippocampal neurogenic niche and neurodegeneration. AIE reduces the neurogenic markers doublecortin (DCX), SRY‐box 2 (SOX2), and Ki‐67 (Macht et al. 2021; Nwachukwu et al. 2022a; Swartzwelder et al. 2019) and increases apoptosis indicated by an increase in cleaved caspase 3 and death receptor 3 (Swartzwelder et al. 2019) within the hippocampus. The effects of AIE on the neurogenic niche have multiple translational implications because hippocampal neurogenesis can affect both hippocampal integrity and memory‐related cognitive functions (Costa et al. 2015). Moreover, compromises of neuronal proliferation, differentiation, and survival are thought to be among the mechanisms of persistent cell death associated with AUDs (Geil et al. 2014).

Promisingly, recent studies have investigated potential treatments and therapeutics to reverse some of these long‐term neurological dysregulations using currently approved and utilized drugs and natural therapies such as regular exercise. For example, previous studies from our laboratory and others have explored the capacity for donepezil (DZ), a cholinesterase inhibitor and a currently prescribed drug to treat Alzheimer's disease, to reverse hippocampal damage due to excessive alcohol consumption (Guo et al. 2015; Swartzwelder et al. 2019). In rodents, evidence has supported DZ as a viable drug to improve neurogenesis in the dentate gyrus (DG) of the hippocampus (Narimatsu et al. 2009), increase Ki‐67 and DCX in Alzheimer's disease models (Mirza et al. 2021), and protect against increases in cleaved caspase 3 during cholinergic depletion (Cutuli et al. 2013). Donepezil inhibits acetylcholinesterase metabolism of acetylcholine, leading to more acetylcholine in the synapse and improved cholinergic transmission (Kumar et al. 2022), and acetylcholine inhibits inflammatory responses by decreasing cytokine secretions and glial activation (Hwang et al. 2010; Kim et al. 2021; Rosas‐Ballina and Tracey 2009). Previously, we found that DZ reversed AIE‐induced decreases in the hippocampal neurogenesis marker, DCX, and increases in the apoptotic marker cleaved caspase 3 and death receptor 3 in adulthood. Moreover, DZ administration decreased the upregulation of neuroinflammatory markers toll‐like receptor 4 (TLR4), receptor for advanced glycation end‐products (RAGE), high mobility group box 1 (HMGB1), and phosphorylated nuclear factor kappa B subunit 1 (pNFκB) (Swartzwelder et al. 2019). However, all previous work was conducted specifically in male rats. With emerging increases in alcohol consumption among female adolescents, it has become imperative to investigate the hypothesis that DZ will reverse such AIE effects in females as well.

To assess the efficacy of adulthood administration of DZ as a treatment to reverse AIE‐induced neurogenic dysregulation, upregulated neuroimmune responses, and neurodegeneration that persists in adulthood, we utilized the AIE model specifically in female rats (Nwachukwu et al. 2022a, 2022b). This study expands upon our most recent work indicating that AIE induced a reduction of neurogenic markers DCX, SOX2, and Ki‐67 in the DG (Nwachukwu et al. 2022a) and caused a significant increase in glial cells in female rats (Nwachukwu et al. 2022b). In this study, we tested the hypothesis that administration of DZ in adulthood can reverse AIE‐induced reduction of neurogenesis as well as the increased glial activation and proinflammatory milieu that contributes to neurodegeneration.

2. Materials and Methods

2.1. Animals and Ethanol Exposure

The methodology and protocols 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). Thirty female Sprague–Dawley rats (Charles River, Raleigh, NC, USA); postnatal day 16 (PND16) arrived at our lab with their dams and were given 7 days to habituate to their environment before weaning, followed by an additional 7 days to acclimate to the vivarium with their cage mates on a reversed 12‐h light: dark cycle before dosing. Two rats were housed per cage with ad libitum access to food and water. Rats were pair‐housed based on treatment groups. Similar to previous studies (Healey et al. 2022b; Nwachukwu et al. 2022a, 2022b), 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 (PND30–52). All animals in the study survived the procedure as the dose is well below the LD50 for rats (Wiberg et al. 1970). This dose was established to mimic the high blood ethanol concentration (BECs) achieved by human adolescents during binge drinking episodes (Ruetsch et al. 2021; Vinader‐Caerols et al. 2017).

AIE exposed rats were administered a total of 14 doses of ethanol on a 2‐days on, 1‐day off, 2‐days on, 2‐days off schedule for a duration of 23 days until all doses were administered based on daily body weights. The adolescent intermittent water (AIW) controls were administered isovolumetric water 18.12 mL/kg on the same schedule. Following dosing, animals were given 25 days to mature into adulthood, and either DZ (2.5 mg/kg) or water was administered 21 days into the maturation period (beginning at PD74) for 4 daily doses (Figure 1) via intragastric gavage (1.88 mL/kg), similar to previous studies (Mulholland et al. 2018; Swartzwelder et al. 2019). All animals were sacrificed two hours after the last DZ dose on PND 78 using rapid decapitation. One hemisphere of the brain was postfixed in 4% (w/v) paraformaldehyde for 24 h, and placed in cryopreserve (1 mM polyvinyl‐pyrrolidone, 50% (v/v) ethylene glycol in 0.2 M phosphate buffer, pH = 7.4) until further processing. Microdissection was used to extract the hippocampus from the other hemisphere for PCR analyses. The experimental groups were as follows: AIW/VEH (n = 7), AIW/DZ (n = 7), AIE/VEH (n = 7), AIE/DZ (n = 7). Separate AIE‐treated rats were used to collect tail blood to reduce the potential for stress related confounds on experimental outcomes (Walker et al. 2013). The mean BEC achieved for female BEC control rats were 218 ± 10.7 mg/dL, 60 min following AIE exposure, congruent with our previous studies (Nwachukwu et al. 2022a, 2022b).

FIGURE 1.

FIGURE 1

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Schematic of adolescent intermittent ethanol (AIE) and donepezil (DZ) treatment.

2.2. Immunohistochemistry

Methods used for IHC protocols were comparable to previous studies (Grifasi et al. 2019; Nwachukwu et al. 2022a, 2022b; Swartzwelder et al. 2019). Concisely, a hemisphere of the brain was sectioned at a series of 1:12 at 40 μm thickness with a Compresstome VF‐300 microtome (Precisionary Instruments Inc., Greenville, NC) (Nwachukwu et al. 2022a, 2022b). A series of phosphate buffered saline (PBS) washes were done after sections had been stored in cryopreserve. After PBS washes, 0.6% H2O2 was used to quench endogenous peroxidases, and to impeding non‐specific antibody binding (0.1% triton‐X and 3% goat serum in 0.1 M PBS), tissue series were incubated in respective primary antibodies and dilutions (Table 1) at 4°C for 48 h. The primary antibodies, DCX, SOX2, Ki‐67, GFAP, Iba‐1, S100B, and cleaved caspase 3 were chosen from commercially available sources specific for neurogenic, astrocytic, and neurodegenerative properties that utilized co‐labeling and western blot techniques for validation. Following primary antibody incubation, sections were washed in a series of PBS washes and then incubated in 1:2000 goat anti‐rabbit secondary antibody (Vector Laboratory, Burlingame, CA) for an hour. Following secondary antibody incubation, sections were subjected to another round of PBS washes before using an ABC peroxidase staining kit (Thermo Scientific, Rockford, IL; 1 h) 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 60 (Thermo Scientific).

TABLE 1.

Primary antibodies used for immunohistochemistry experiments.

Antibodies Isotype Purification Dilution Source
DCX Rabbit IgG Polyclonal 1:400 Abcam, Waltham, MA; ab18723
Ki‐67 Rabbit IgG Monoclonal 1:200 Thermo Fisher Scientific, Waltham, MA; MA5‐14520
SOX2 Rabbit IgG Polyclonal 1:200 Millipore Sigma, Burlington, MA; AB5603
GFAP Rabbit IgG Polyclonal 1:2500 Millipore; Billerica, MA; AB5804
S100B Rabbit IgG Polyclonal 1:500 Thermo Fisher Scientific, Waltham, MA; PA5‐78161
Iba‐1 Rabbit IgG Polyclonal 1:1000 Fujifilm Wako Pure Chemical Corporation, Osaka, Japan, 019‐19741
Cleaved Caspase 3 Rabbit IgG Polyclonal 1:400 Cell signaling Technology, Danvers, MA; #9661
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2.3. Immunohistochemistry Quantification

The photomicrographs of the hippocampal subregions were taken using a 10× and 40× objectives on a B120 microscope (AmScope, Irvine, CA) with an attached MU500‐HS AmScope digital camera, as previously described (Grifasi et al. 2019; Nwachukwu et al. 2022a, 2022b). Experimenters were blinded to treatment groups as animals were assigned a random number to analyze the data. Measurements for immunohistochemistry experiments were analyzed in adulthood following AIE exposure to determine the persisting effect of adolescent exposure following a period of discontinuation from ethanol. Automated cell counts were assessed using the open‐source software QuPath 0.2.3 (Bankhead et al. 2017). SOX2, DCX, Ki‐67, and cleaved caspase 3 were assessed solely in the DG because this subregion is the site for neurogenesis. The DG was individually traced on each photomicrograph between Bregma −2.30 mm and −4.52 (Paxinos and Watson 2009). Additionally, GFAP, S100B, and Iba‐1 were assessed in the cornu ammonis 1 (CA1), cornu ammonis 2/3 (CA2/3), and the DG region because glia are more ubiquitously located throughout the hippocampus, where each subregion was individually traced on each photomicrograph between Bregma −2.30 mm and −4.52. A user‐defined threshold for DAB+ pixels and pixels per cell was used to estimate the number of GFAP, Iba‐1, and Sox‐2+ cells. However, DCX+ cells were manually counted due to the clustering nature and density of the complex cell population. Likewise, due to the morphology of Ki‐67+ cells via proliferation, positive cells were manually counted to decrease discrepancies. All cell counts were expressed as cells/mm2. However, cleaved caspase 3 and S100B were assessed using immunoreactivity and were expressed as percent area. Animals with fewer than 4 usable hippocampal sections were excluded from the analysis.

2.4. Quantitative Reverse Transcription Polymerase Chain Reaction

RNA was extracted from the hippocampus using the Trizol (Invitrogen Corporation, Carlsbad, CA) method following the manufacturer's instructions following homogenization using the QSonica CL‐786 sonicator (Newtown, CT), similar to our previous publication (Grifasi et al. 2019). A Qubit 3.0 Fluorometer was used to quantify the total RNA in each sample, which was then normalized so that 1 μg of RNA was reversed transcribed to cDNA. Primers were purchased from ThermoFisher Life Technologies for the measurement of glial transcripts, S100B (Rn04219408_m1), GFAP (Rn01253033_m1), Itgam (Rn00709342_m1) and Aif‐1 (Rn03993468_g1), with Ppia (Rn00690933_m1) used as the endogenous control. Aif‐1 is another name for Iba‐1, but an additional gene, Itgam, was used to understand microglia activity, as its expression is more representative of inflammatory microglial phenotypes (Jurga et al. 2020). The ddCT method was employed such that measurements were normalized to the water‐vehicle control group and expressed as fold change.

2.5. Statistical Analysis

All data were graphed and analyzed using GraphPad Prism 9.5.0 (GraphPad Software Inc. La Jolla, Ca). Data points were evaluated using two‐way analyses (AIE × DZ) of variance (ANOVAs) followed by Bonferroni test post hoc analyses only if a significant interaction was indicated. Effects were considered significant if p < 0.05.

3. Results

3.1. AIE Produced Long‐Term Reduction of DCX Neurons in the DG, and DZ Treatment in Adulthood Ameliorated That Reduction

The potential ameliorating effects of DZ on the AIE‐induced reduction of DCX‐positive cells were assessed utilizing manual cell counts (Figure 2). There was a main effect of AIE [F (1,20) = 34.53, p < 0.0001] but not DZ [F (1,20) = 4.22, p = 0.0533], as well as an interaction [F (1,20) = 9.02, p = 0.0070] on DCX cell number in the DG (Figure 2E). A post hoc Bonferroni analysis revealed that the AIE/DZ had more DCX+ cells than AIE/VEH, suggesting that DZ was able to ameliorate AIE‐induced DCX reduction.

FIGURE 2.

FIGURE 2

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Photomicrographs suggest that doublecortin+ (DCX) cells are decreased in the subgranular zone (SGZ) of the dentate gyrus (DG) hippocampal subregion of female rats following AIE exposure (C) compared to their adolescent intermittent water (AIW) control groups (A) and restored by DZ in AIE‐exposed (D) but not AIW (B) rats. A two‐way ANOVA of DCX+ cell counts indicated that AIE decreased the number of DCX+ cells but was ameliorated by DZ (E). Scale bar = 20 μm; *p < 0.05 AIE‐Vehicle versus AIE‐DZ.

3.2. AIE Decreased KI‐67+ Cells in the DG and DZ Restored KI‐67 Neuronal Loss in Adulthood

The effects of DZ on AIE‐induced reduction of KI‐67+ cells were assessed utilizing manual cell counts to determine neurogenesis proliferation (Figure 3). A two‐way ANOVA (AIE × DZ) showed a main effect of AIE [F (1,21) = 8.24, p = 0.009] and a main effect of DZ [F (1,21) = 12.52, p = 0.002], as well as an interaction [F (1,21) = 27.39, p < 0.0001] (Figure 3E). Post hoc Bonferroni analysis indicated that DZ restored Ki‐67+ cell numbers following AIE treatment.

FIGURE 3.

FIGURE 3

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Photomicrographs indicate Ki‐67+ cells are decreased in the SGZ of the DG hippocampal subregion of female rats following AIE exposure (C) compared to their AIW control groups (A) and restored by DZ in AIE‐exposed (D) but not AIW (B) rats. A two‐way ANOVA of Ki‐67+ cell counts revealed that AIE decreased the number of Ki‐67+ cells but was ameliorated by DZ (E). Scale bar = 20 μm; *p < 0.05 AIE‐Vehicle versus AIE‐DZ.

3.3. AIE Decreased SOX2+ Cells in the DG and DZ Restored SOX2 Neuronal Loss in Adulthood

The effects of DZ on AIE‐induced reduction of SOX2+ cell numbers were assessed utilizing automated cell counts to determine stem cell neurogenesis (Figure 4). A two‐way ANOVA (AIE × DZ) revealed main effects of AIE [F (1,20) = 10.18, p = 0.046] and DZ [F (1,20) = 9.44, p = 0.006] as well as their interaction [F (1,20) = 13.83, p = 0.001] (Figure 4E). Post hoc Bonferroni analysis indicated that AIE‐induced reduction of SOX2 was attenuated by DZ administration compared to their controls.

FIGURE 4.

FIGURE 4

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Photomicrographs suggest SOX2+ cells are decreased in the DG hippocampal subregion of female rats following adolescent intermittent ethanol (AIE) exposure (C) compared to their AIW control groups (A) and restored by donepezil (DZ) in AIE‐exposed (D) but not AIW (B) rats. Post hoc analyses revealed that AIE decreased the number of SOX2+ cells but was ameliorated by DZ (E). Scale bar = 20 μm; *p < 0.05 AIE‐Vehicle versus AIE‐DZ; hilus of the DG is indicated within the white dashes.

3.4. AIE Increased GFAP+ Cells in all Subregions of the Hippocampus and DZ Reduced Astrogliosis in Only the CA2/3 Subregion in Adulthood

The efficacy of DZ on AIE‐induced GFAP+ cell counts was assessed utilizing automated cell counts as a correlate of astrocytic activation (Figure 5). In the DG subregion, a two‐way ANOVA (AIE × DZ) showed a main effect of AIE [F (1,21) = 4.70, p = 0.042]; however, there was no interaction [F (1,21) = 0.71, p = 0.41] or main effect of DZ [F (1,21) = 0.018, p = 0.89] on the number of GFAP+ cells (Figure 5E). Additionally, a two‐way ANOVA indicated that there was a main effect of AIE [F (1,21) = 33.29, p = 0.004] but no effect of DZ [F (1,21) = 0.004, p = 0.95] nor interaction [F (1,21) = 0.17, p = 0.68] in the CA1 subregion of the hippocampus (Figure 5F). Finally, there was a main effect of AIE [F (1,21) = 10.6, p = 0.004] and DZ [F (1,21) = 6.60, p = 0.018], as well as an interaction [F (1,21) = 5.24, p = 0.033] on astrocyte number in the CA2/3 (Figure 5G). A post hoc Bonferroni analysis revealed that the AIE/DZ had fewer GFAP+ cells than AIE/VEH, suggesting that DZ was able to ameliorate AIE‐induced increases in GFAP+ cells.

FIGURE 5.

FIGURE 5

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Photomicrographs suggest glial fibrillary acidic protein (GFAP+) cells are increased in the CA2/3 subregion of female rats following AIE exposure (C) compared to their AIW control groups (A) but effects are reversed with DZ (D). A two‐way ANOVA of GFAP+ cell counts indicated that AIE increased the number of GFAP+ cells in the DG (E), CA1 (F), and CA2/3, with a DZ reversal in this subregion (G). Scale bar = 20 μm; *p < 0.05 Vehicle versus DZ, #p < 0.05 AIW versus AIE; *p < 0.05 AIE‐Vehicle versus AIE‐DZ.

3.5. AIE Increased S100B Specifically in the DG Which Was Reversed in Adulthood by DZ

S100B is a calcium‐binding protein associated with astrocyte activation and maturity, but its localization and function within astrocytes differ from GFAP (Janigro et al. 2022; Raponi et al. 2007). A two‐way ANOVA indicated main effects of AIE [F(1,19) = 4.86, p = 0.04] and DZ treatment [F(1,19) = 7.62, p = 0.01] as well as an interaction [F(1,19) = 18.29, p < 0.001]. Posthoc Bonferroni's multiple comparisons tests suggested that AIE led to an increase in S100B immunoreactivity that DZ ameliorated these effects (Figure 6). The effect of both DZ and AIE on S100B immunoreactivity appeared to be unique to DG (Figure 6E) as no main effects or interactions were seen for the CA1 (AIE [F(1,19) = 2.76, p = 0.11]; DZ [F(1,19) = 0.79, p = 0.38]; interaction [F(1,19) = 4.17, p = 0.06]) or CA/23 (AIE [F(1,20) = 0.34, p = 0.56]; DZ [F(1,20) = 1.53, p = 0.23]; interaction [F(1,20) = 0.10, p = 0.75]) regions.

FIGURE 6.

FIGURE 6

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Photomicrographs suggest S100B+ immunoreactivity is increased in the DG subregion of female rats following AIE exposure (C) compared to their AIW control groups (A, B) but effects are reversed with DZ (D). A two‐way ANOVA of S100B+ immunoreactivity indicated that AIE increased the percent area of S100B in the DG, with a DZ reversal in this subregion (E). However, neither AIE nor DZ impacted S100B in the CA1(F) or CA2/3 (G). Scale bar = 20 μm; *p < 0.05 Vehicle versus DZ, #p < 0.05 AIW versus AIE; *p < 0.05 AIE‐Vehicle versus AIE‐DZ.

3.6. AIE Caused an Increase in the Number Iba‐1+ Cells in the DG That Was Reduced in Adulthood by DZ

As expected, AIE led to an increase in the number of microglia in the DG (Figure 7) as a two‐way ANOVA (AIE × DZ) showed a main effect of AIE [F(1,23) = 6.28, p = 0.02] and an interaction was detected [F(1,23) = 7.41, p = 0.012] but not a main effect of DZ [F(1,23) = 3.78, p = 0.06] (Figure 7E). Likewise, in CA1, a two‐way ANOVA (AIE × DZ) showed a main effect of AIE [F(1,23) = 8.40, p = 0.008] but not DZ [F(1,23) = 0.054, p = 0.82] nor any interaction between the variables [F(1,23) = 2.00, p = 0.17] (Figure 7F). Finally, in the CA2/3 region, there were no main effects (AIE [F(1,23) = 1.21, p = 0.28]; DZ [F(1,23) = 2.83, p = 0.11]) nor any interaction [F(1,23) = 1.21, p = 0.28] (Figure 7G).

FIGURE 7.

FIGURE 7

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Photomicrographs suggest Ionized calcium‐binding adapter molecule 1 (Iba‐1+) cells are increased by ethanol in the DG (C) compared to the control (A). Importantly, DZ reduces this increase in microglia number (D). A two‐way ANOVA of Iba1+ cell counts further supported that AIE increased the number of Iba‐1+ cells in the DG with a DZ reversal in this subregion (E), but no effects in the CA1 (F) or CA2/3 (G) subregions were observed. Scale bar = 20 μm; *p < 0.05 AIE‐Vehicle versus AIE‐DZ.

3.7. AIE Caused Persistent Increases in Cleaved‐Caspase 3 Immunoreactivity in the DG Hippocampal Subregion

The effects of DZ on AIE‐induced cleaved caspase 3+ increased immunoreactivity were assessed utilizing densitometry to assess apoptosis (Figure 8). A two‐way ANOVA (AIE × DZ) showed a main effect of AIE [F (1,21) = 8.01, p = 0.01] and a main effect of DZ [F (1,21) = 6.85, p = 0.02], no interaction was detected [F (1,21) = 1.68, p = 0.21] (Figure 8E). Ethanol increased cleaved caspase 3+ immunoreactivity, while DZ decreased cleaved caspase 3+ cells independent of ethanol exposure.

FIGURE 8.

FIGURE 8

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Photomicrographs reveal that cleaved caspase 3+ immunoreactivity is significantly increased in the DG hippocampal subregion of female rats following AIE exposure (C) compared the AIW group (A) DZ (B, D) reduced cleaved caspase 3+ immunoreactivity independently compared with vehicle (A, C). A two‐way ANOVA of cleaved caspase 3+ IR revealed that AIE increased the number of cleaved caspase 3+ pixels and that DZ reduces cleaved caspase immunoreactivity (E). Scale bar = 20 μm; #p < 0.05 AIW versus AIE; *p < 0.05 Vehicle versus DZ hilus of the DG is indicated within the white dashes.

3.8. AIE Only Increased GFAP mRNA and DZ Reduced Glial Gene Expression Independently of Ethanol

To follow up on the increases in glial associated proteins observed in IHC, mRNA levels of astrocytic genes, GFAP and S100B, as well as microglial genes, Itgam and Aif‐1 were measured in the hippocampus. GFAP was the only gene that was significantly increased by AIE according to a two‐way ANOVA which showed a main effect of AIE [F (1,24) = 16.93, p < 0.001]. DZ [F (1,24) = 4.49, p = 0.048] independently decreased GFAP expression in the DG, but there was no interaction between the two variables [F (1,24) = 3.01, p = 0.10] (Figure 9A). Two‐way ANOVAs indicated that AIE did not lead to significant changes in S100B [F (1,24) = 0.11, p = 0.48], Aif‐1 [F (1,24) = 0.64, p = 0.43] or Itgam [F (1,24) = 0.04, p = 0.85] with no interactions between DZ and AIE (S100B [F (1,24) = 0.005, p = 0.48]; Aif‐1 [F (1,24) = 0.64, p = 0.43]; Itgam [F (1,24) = 0.04, p = 0.85]); however, DZ did independently reduce (S100B [F (1,24) = 6.255, p = 0.0196] and Aif‐1 [F (1,24) = 4.671, p = 0.04309]) but not Itgam [F (1,24) = 3.04, p = 0.09] (Figure 9).

FIGURE 9.

FIGURE 9

Open in a new tab

GFAP mRNA was significantly upregulated after AIE (A) but no effect of AIE exposure was observed in any of the other glial genes, including S100B (B), Aif‐1 (C), and Itgam (D). Independent of ethanol exposure, DZ decreased the mRNA fold change of GFAP (A), S100B (B), and Itgam (D). #p < 0.05 AIW versus AIE; *p < 0.05 Vehicle versus DZ.

4. Discussion

Emerging studies have begun to explore possible preventative and treatment options for the effects of adolescent alcohol use. Several drugs in current clinical use for other indications have been explored. These include the anticonvulsant drug gabapentin (Anton et al. 2020; Healey et al. 2022a; Healey et al. 2020; Li et al. 2022; Swartzwelder, Park, and Acheson 2017), the opioid antagonist nalmefene (Montesinos et al. 2017; Tournier et al. 2021), the lipid‐lowering agent fenofibrate (Villavicencio‐Tejo et al. 2021), and the anti‐inflammatory agent indomethacin (Collins et al. 1985; Deschamps et al. 2022; Vetreno and Crews 2018; Vetreno et al. 2018). The anticholinesterase DZ is currently used for cognitive enhancement in early dementia and has been suggested as a potential therapeutic against neuroimmune responses and degeneration in studies of stroke (Madani Neishaboori et al. 2021; Song et al. 2021) and schizophrenia (Li et al. 2018). It has also been shown to ameliorate the effects of AIE on multiple measures of neural function, including the elevation of neuroimmune markers (Mulholland et al. 2018; Swartzwelder et al. 2019), but those studies did not include female subjects. The present study aimed to determine the reversal effects of DZ on the AIE‐induced neuroimmune, neurogenic, and apoptotic responses in the hippocampus, specifically in female rats. The major findings from this study are (1) AIE increased markers of cell death, microglial activation, and astrogliosis; and decreased markers of neurogenesis in the hippocampal formation of adult female rats, and (2) treatment with DZ in adulthood reversed all of those AIE effects, some with hippocampal subregion specificity.

The reduction of neurogenic markers DCX, Ki‐67 and SOX2 in the DG subregion of the female hippocampus, are consistent with previous findings from our lab (Nwachukwu et al. 2022a) and others (Swartzwelder et al. 2019) that used male rats. Additionally, these data dovetail with other studies that have shown that chronic adolescent binge drinking impacts neurogenesis (Broadwater et al. 2014; Crews et al. 2006; Liu and Crews 2017; Macht et al. 2021; Swartzwelder et al. 2019). The present study adds to this body of work by demonstrating that sub‐chronic post‐AIE administration of DZ in adulthood is sufficient to reverse the reduction of neurogenesis in the adult hippocampus of female rats denoted by an increase in DCX, Ki‐67, and SOX2 of the rats exposed to AIE. To our knowledge no other studies have investigated the efficacy of DZ on neurogenesis in the post‐AIE adult female rodent brain. DCX is known to play a role in neuronal migration and differentiation (Francis et al. 1999), while SOX2 is involved in stem cell differentiation (Kishi et al. 2000), and Ki‐67 in neuronal proliferation (Burger et al. 1986). Dysregulation of any of these markers would impact and hinder neurogenesis (Liu and Crews 2017), which ultimately could lead to adult cognitive deficits and neurodegeneration (Geil et al. 2014; Vetreno and Crews 2015). Taken together the observed restorative effects of DZ on all three neurogenic markers suggest that DZ can reinstate multiple features of the neurogenic process that are compromised by AIE exposure in the hippocampal formation, with possible therapeutic implications for both cognitive and affective functioning after adolescent alcohol exposure.

In this study, we were able to replicate, in female rats, our previous findings in males that AIE exposure induces persisting increases in GFAP+ cells across all hippocampal subregions and Iba‐1+ cells, specifically in the DG subregion (Nwachukwu et al. 2022b; Risher et al. 2015). This study also added a second marker of astrocyte activation, S100B, which further indicated that AIE effects on astrocytes are long lasting. However, there were some regional differences in AIE effects on S100B and GFAP. More specifically, AIE‐induced increases in astrocyte activation based on S100B were only reflected in the DG compared with GFAP, which was consistently increased in all hippocampal subregions after AIE. In addition to replicating those findings, we observed a reversal of those AIE‐induced effects after sub‐chronic administration of DZ during adulthood. The fact that DZ is a safe and commonly used medication for other indications is promising in the context of the possible development of ameliorative treatments for the enduring sequelae of adolescent alcohol use. While we only observed an increase in GFAP mRNA in adulthood after AIE, DZ significantly decreased Aif‐1, GFAP, and S100B mRNA levels, further supporting the therapeutic potential of DZ not only in AUD but also in other diseases associated with glial dysregulation. Similar promise has been observed with the lipid‐regulating medication, fenofibrate, which has been shown to mitigate GFAP hyperexpression during ethanol withdrawal in adolescent rodents (Villavicencio‐Tejo et al. 2021). Similarly, physical exercise has been shown to reverse Iba‐1+ cell increases in the dorsal raphe nucleus following AIE exposure (Vetreno et al. 2017). Such studies underscore the fact that, although many AIE effects persist into adulthood, they are also reversible with treatments ranging from behavioral alterations to safe and commonly used medications. The fact that DZ‐induced reversal of AIE effects is observed in both sexes amplifies the translational value of these emerging sets of findings, as does the present finding that DZ diminishes gliosis independent of AIE exposure. Furthermore, while no significant changes were observed in S100B and Aif‐1 mRNA fold change between AIW and AIE groups, the discernible reduction in these glial genes by DZ administration provides additional rationale for donepezil's ability to ameliorate AIE‐induced gliosis. Outside of our previous work (Swartzwelder et al. 2019) minimal literature exists on the efficacy of DZ as a treatment for neuroimmune dysregulation consequent to alcohol abuse. Therefore, the present studies underscore both the therapeutic potential of DZ and the need to continue to explore it as a candidate for multiple possible applications related to alcohol and other substance use.

We have previously reported an increase in the apoptotic marker cleaved caspase 3 in the hippocampus of male rats following AIE, which was reversed by sub‐chronic DZ treatment in adulthood (Swartzwelder et al. 2019). The present study expanded that finding and determined that similar effects occur in female rats, although the effects of DZ in females were not specific to alcohol. Galantamine, another cholinesterase inhibitor used to treat Alzheimer's patients, has also been shown to prevent and reverse the activation of cleaved caspase 3 induced by AIE when administered in adulthood (Macht et al. 2021). Interestingly, the persisting increase of cleaved caspase 3 effects is observed after adolescent, but not adult, intermittent ethanol exposure (Broadwater et al. 2014). Similar differential effects of repeated ethanol exposure during adolescence, compared to adulthood, have been observed in behavioral (White et al. 2000, 2002; Risher et al. 2015) and electrophysiological (Fleming et al. 2012) studies. Such findings highlight the susceptibility of the adolescent brain to respond to injuries and insults, including those produced by repeated ethanol exposure. It is important to note that, despite the current observation of elevated cleaved caspase 3 in females and previously seen effects in males (Swartzwelder et al. 2019), prior results showed no changes in the fluoro‐jade‐C staining in AIE females (Nwachukwu et al. 2022b). This could be due to substantive cessation of active neurodegeneration during the 25‐day period between the end of AIE and the sacrifice of animals in adulthood. Thus, it may be that AIE produces a persistent increase in the propensity toward apoptotic cell death in the absence of marked ongoing neurodegeneration. Importantly, this study further supports the role of DZ in reducing apoptosis and cleaved caspase 3 (Li et al. 2023; Ongnok et al. 2021) even in the absence of alcohol. This idea was particularly supported by the qRT‐PCR results, which indicated that DZ reduced three of the four glial genes even if there were not consistent alcohol‐related effects.

One important limitation of the current study is the inability to do a direct comparison of the effects of donepezil on the reversal of AIE‐related effects in glial, neurogenic, and neurodegenerative processes across the sexes. Our previous work established that for many of the glial and neurogenic maladaptations, the impact of AIE was very similar between males and females, with one distinct difference: female rats had more persisting increases in microglial activation after AIE compared with ethanol‐exposed males (Nwachukwu et al. 2022a, 2022b). As such, the current study only examined the effects of DZ in females; however, our previous study examined the effects of DZ on AIE reduction in DCX and cleaved caspase 3 in males (Swartzwelder et al. 2019). Due to differences in the methodology between the studies, a direct statistical comparison is not plausible. However, DZ increased DCX in males by 164% (Swartzwelder et al. 2019) and in females by 150% after AIE. Likewise, AIE increased cleaved‐caspase 3 in males and females across the two studies. Although the decrease in cleaved‐caspase 3 from DZ administration observed herein was not specific to alcohol alone, the extent of DZ effects was almost twice as much in females (50%) in the current study compared with our previous publications in males (26%). The impact of sex on the therapeutic potential of immune modulators on alcohol‐induced neuropathological consequences remains to be determined after AIE; however, clinical and preclinical evidence suggests that the amelioration of behavioral and neurologic maladaptations by phosphodiesterase inhibitors is equivalent across sex (Grigsby et al. 2023; Grodin et al. 2021; Jimenez Chavez et al. 2021; Meredith et al. 2022). To fully understand sex as a biological variable in DZ's effectiveness, it is critical to include this factor in future studies (Nwachukwu et al. 2023), especially as there are still questions whether donepezil is more effective in men or women with Alzheimer's Disease (Canevelli et al. 2017).

The present findings expand on the growing literature by highlighting the effectiveness of DZ as a reversal drug for AIE‐induced neurological deficits in female animals and underscore the cholinergic system as a therapeutic target of interest for reducing AIE‐induced neuroimmune and neurobiological responses, including decreased neurogenesis, increased astrogliosis, and apoptosis in the hippocampus. More studies are needed to explore the behavioral maladaptations that may be caused by AIE‐induced neurobiological and neuroimmune responses, such as deficits in learning, memory, executive function, and affective regulation, and whether DZ can restore such deficits.

Ethics Statement

All procedures used in this study were approved by the Duke University Institutional Animal Care and Use Committee (Protocol Registry Number A159‐18‐07) and followed the guidelines for the Care and Use of Laboratory Animals.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank Dr. Diego Correia, Amaya Jackson, Taby Michel, and Favor Asabor for technical assistance in experimentation.

Funding: This work was supported by the National Institute on Alcohol Abuse and Alcoholism (U54AA019765, U54AA030451, R25AA030409, U01AA019925, U01AA019925S2) and the National Institute on General Medical Sciences (SC1GM139696).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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