Resistance of postnatal hippocampal neurogenesis to alcohol toxicity in a third trimester-equivalent mouse model of gestational alcohol exposure

Abstract

Background:

The adult hippocampal dentate is comprised of both developmentally-generated dentate granule cells (dDGCs) and adult-generated dentate granule cells (aDGCs), which play distinct roles in hippocampal information processing and network function. EtOH exposure throughout gestation in mouse impairs the neurogenic response to enriched environment (EE) in adulthood, although the basal rate of adult neurogenesis under standard housing (SH) is unaffected. Here, we tested whether the production and/or survival of either dDGCs or aDGCs are selectively impaired following exposure of mice to EtOH vapors during early postnatal development (human 3^rd trimester-equivalent), and whether this exposure paradigm leads to impairment of EE-mediated dentate neurogenesis in adulthood.

Methods:

All experiments were performed using NestinCreER^T2:tdTomato bitransgenic mice, which harbor a tamoxifen-inducible tdTomato (tdTom) reporter for indelible labeling of newborn hippocampal DGCs. We exposed all mice to EtOH vapor or room air (Control) for 4 hours (hr) per day from postnatal day (PND) 3 through PND 15. This paradigm resulted in a mean daily post-exposure blood EtOH concentration (BEC) of ~160 mg/dl. One cohort of neonatal mice received a single injection of tamoxifen at PND 2 and was sacrificed at either PND 16 or PND 50 to assess the impact of EtOH exposure on the production and long-term survival of dDGCs born during the early postnatal period. A second cohort of mice received daily injections of tamoxifen at PND 35-39 to label aDGCs, and was exposed to SH or EE for 6 weeks prior to sacrifice.

Results:

Early postnatal EtOH exposure had no statistically significant effect on the production or survival of tdTom⁺ dDGCs, as assessed at PND 16 or PND 50. Early postnatal EtOH exposure also had no effect on the number of tdTom+ aDGCs under SH conditions. Furthermore, early postnatal EtOH exposure had no significant impact on the adult neurogenic response to EE.

Conclusions:

Both early postnatal dentate neurogenesis and adult dentate neurogenesis, as well as the adult neurogenic response to EE, are surprisingly resistant to early postnatal EtOH vapor exposure in mice.

Introduction

FASD is a significant public health concern as the leading preventable cause of intellectual and neurodevelopmental disability (Mattson et al., 1999, Guerri et al., 2009, Riley and McGee, 2005), with an estimated prevalence of 2-5% in school-age children within the United States (May et al., 2014). Approximately 1 in 10 women report alcohol consumption at some stage of pregnancy despite widespread efforts at public awareness (Tan et al., 2015). Therefore, it is important to identify therapeutic targets and strategies that can mitigate the neurodevelopmental disabilities after FASD has been clinically diagnosed (Kodituwakku, 2010). Although developmental EtOH exposure exerts teratogenic effects in many brain areas, impaired function of the hippocampus is particularly well-documented in both clinical FASD and preclinical models (Autti-Ramo et al., 2002, Berman and Hannigan, 2000, Hamilton et al., 2003, Petrelli et al., 2018, Farr et al., 1988, Savage et al., 2002, Sutherland et al., 1997). Abnormalities in both clinical FASD and animal models include reduced hippocampal volume and deficits in hippocampal-dependent functions such as learning, memory, and mood regulation (Autti-Ramo et al., 2002, Hamilton et al., 2003, Kodituwakku, 2007, Parnell et al., 2009)

Postnatal hippocampal neurogenesis, characterized by the continued production of DGCs throughout life, represents one potential therapeutic target for mitigating certain neurological outcomes in FASD. Although the majority of DGCs are produced during brain development, continued production of newborn DGCs throughout life plays a critical role in the promotion of flexible learning and adaptive behavioral responses to cognitive and emotional challenge (Anacker and Hen, 2017, Clelland et al., 2009, Danielson et al., 2016, Denny et al., 2012, Nakashiba et al., 2012, Swan et al., 2014). Impairment of dentate neurogenesis in experimental animals during young adulthood leads to dysregulation of cognition, mood and stress resilience, which are aspects of mental health often affected in clinical FASD (Hellemans et al., 2010, O'Connor et al., 2002, Pei et al., 2011, Streissguth and O'Malley, 2000). Importantly, postmortem analysis has also demonstrated lifelong dentate neurogenesis in human (Boldrini et al., 2018, Moreno-Jimenez et al., 2019, Spalding et al., 2013). Although it is unknown whether impaired neurogenesis occurs in clinical FASD, reductions in hippocampal volume and impaired temporal lobe network function that correlate with hippocampal-related behavioral deficits in FASD are consistent with this possibility (Autti-Ramo et al., 2002, Riikonen et al., 1999, Sowell et al., 2007, Willoughby et al., 2008).

Preclinical rodent studies have shown that developmental EtOH exposures lead to long-lasting deficits in postnatal dentate neurogenesis, which vary in severity depending upon timing, dose and route of administration (Choi et al., 2005, Ieraci and Herrera, 2007, Kajimoto et al., 2013, Klintsova et al., 2012, Klintsova et al., 2007, Gil-Mohapel et al., 2014). We have previously demonstrated that exposure to relatively moderate levels of EtOH throughout gestation in mice (~80-120 mg/dl maternal BEC) markedly impairs the neurogenic response to EE in adulthood, without altering basal rates of neurogenesis under SH conditions (Choi et al., 2005, Kajimoto et al., 2013). These effects are not due to impaired progenitor proliferation or size of the progenitor pool, but are most likely due to impaired activity-dependent survival and incorporation of early postmitotic DGCs into the existing hippocampal circuitry under conditions of EE (Choi et al., 2005, Kajimoto et al., 2013). Impaired EE-mediated neurogenesis in this model of moderate gestational EtOH exposure correlates with impaired A-B contextual fear-discrimination learning (Kajimoto et al., 2013), a behavioral task previously demonstrated by us and others to be dependent upon the activity of aDGCs (Gustus et al., 2018, Kheirbek et al., 2012, Niibori et al., 2012, Tronel et al., 2015). Interestingly, gestational EtOH exposure also results in a compensatory increase (~2-fold) in the frequency of spontaneous excitatory postsynaptic currents within surviving aDGCs under conditions of EE, as assessed by patch clamp recordings in hippocampal slice preparations, concomitant with impaired EE-mediated dendritic branching and excitatory synaptic activity in older pre-existing, dDGCs (Kajimoto et al., 2016). These observations suggest that the adult hippocampal circuitry in gestationally exposed mice does not respond appropriately to EE stimulation. In terms of clinical FASD, these findings imply that exposure to even moderate levels of EtOH during gestation may limit the ability of individuals to optimally adapt to enriched social and physical experiences, and possibly limit their ability to respond optimally to certain types of behavioral training therapy.

The long-term impact of gestational EtOH exposure on adult hippocampal dentate neurogenesis in mice is surprising, given that ~85% of all DGCs are generated after birth in rodents, with peak rates of neurogenesis occurring between PND 5-7 (Altman and Bayer, 1990, Bayer, 1980), after the cessation of gestational EtOH exposure. dDGCs in rodents born during this early postnatal period arise from rapidly proliferating progenitors that originate in the embryonic dentate neuroepithelium (Berg et al., 2019, Li et al., 2013). Following formation of the dentate granule cell layer, these progenitors become restricted to the dentate subgranular zone (SGZ) by PND 14, where they transition into quiescent radial glial cells that continue to produce aDGCs at a much slower rate throughout life (Berg et al., 2019, Mathews et al., 2010, Nicola et al., 2015). Although human dentate neurogenesis peaks during mid-gestation with the formation of the dentate gyrus mostly completed by birth (Arnold and Trojanowski, 1996), recent studies on postmortem brains support continued dentate neurogenesis throughout the human lifespan (Moreno-Jimenez et al., 2019, Boldrini et al., 2018, Snyder, 2019, Gage, 2019, Tobin et al., 2019), but see (Sorrells et al., 2018). Thus, the adult hippocampal dentate in both humans and rodents is comprised of a heterogeneous population of dDGCs and aDGCs of various ages. Importantly, accumulating evidence indicates that DGCs play distinct roles in dentate information processing and network function depending upon their cellular ontogenetic age (Luna et al., 2019). In the present study, we asked whether exposure to EtOH vapor during the period of peak dentate neurogenesis in the early postnatal period (~human 3^rd trimester equivalent for brain development) impairs the production and/or long-term survival of dDGCs, and/or the production of aDGCs under conditions of SH or EE. We utilized vapor chambers to deliver EtOH to mouse pups during the first two weeks of postnatal development at a dose known to result in acute oligodendrocyte loss and persistent white matter injury (Newville et al., 2017). We utilized a well-characterized Cre-loxP genetic labeling approach in NestinCreER^T2:tdTomato bitransgenic mice to identify newborn DGCs. These mice harbor a tamoxifen- inducible tdTomato reporter for indelible labeling of newborn DGCs in vivo (Cunningham et al., 2017, Gustus et al., 2018). Surprisingly, we found that moderate EtOH exposure during the early postnatal period impacts neither the production nor the survival of dDGCs, and has no effect on the production of aDGCs or the ability to mount a neurogenic response to EE in adulthood. These findings demonstrate resistance of early postnatal neurogenesis to levels of EtOH exposure that disrupt other aspects of postnatal brain development (e.g., oligodendrogenesis and white matter integrity) and further indicate that the impact of developmental EtOH exposure on adult dentate neurogenesis is highly dependent on timing, dose and/or route of alcohol administration.

Materials and Methods

Animals

Mice used in this study were obtained from breeding colonies maintained in the University of New Mexico Health Sciences Center Animal Research Facility. Mice were housed under reverse 12-hr dark/12-hr light cycle (lights off at 0800 hr; lights on at 2000 hr) in temperature and humidity controlled rooms with access to ad libitum food and water. All experiments were performed using Nestin-CreER^T2: tdTomato bitransgenic mice maintained on a C57Bl/6 genetic background with homozygosity at both transgene loci (Li et al., 2010a). These bitransgenic mice harbor a tamoxifen inducible CreER^T2 fusion protein under control of the nestin promoter (Jackson Stock No. 016261) (Lagace et al., 2007) and a loxP-flanked transcriptional STOP cassette upstream of the tdTom reporter allele at the Rosa26 locus (Jackson Stock No.007909) (Madisen et al.,2010). Tamoxifen administration results in Cre-mediated recombination and tdTom reporter expression within nestin⁺ hippocampal progenitors and downstream progeny (Cunningham et al., 2017, Lagace et al., 2007, Li et al., 2010b, Newville et al., 2017). All animal procedures were approved by the University of New Mexico Health Sciences Center Institutional Animal Care and Use Committee guidelines per the NIH Animal Welfare Regulations and Public Health Service Policy on Human Care and Use of Laboratory Animals.

Genotyping

Verification of genotype was completed using a PCR assay. Genomic DNA (gDNA) was isolated from tail snips immersed in DirectPCR tail lysis buffer (Viagen cat#102-T) and Proteinase K 20mg/mL (Viagen cat#501-PK) at 55°C overnight. This was followed by 45 minutes at 85°C to deactivate the proteinase K enzyme prior to PCR. Cre PCR mixture contains 50% Hot Star Taq Master Mix (Qiagen Cat No.203445), 3.6% Primers (IDT), 44.4% RNase-Free H₂O (Qiagen 1012888), and 2% gDNA. Rosa 26 tdTomato PCR mixture contains 50% Hot Star Taq Master Mix (Qiagen Cat No.203445), 6% Primers (IDT), 40% RNase-Free H₂O (Qiagen 1012888), and 4% gDNA. The following primer sequences were used to identify the presence of Cre and Rosa 26 tdTomato : Cre Forward 5’GCG GTC TGG CAG TAA AAA CTA TC 3’, Cre Reverse 5’ GTG AAA CAG CAT TGC TGT CAC TT 3’, Rosa Wildtype Forward 5’ AAG GGA GCT GCA GTG GAG TA 3’, Rosa Wildtype Reverse 5’ CCG AAA ATC TGT GGG AAG TC 3’, Rosa Mutant Forward 5’ CTG TTC CTG TAC GGC ATG G 3’, and Rosa Mutant Reverse 5’ GGC ATT AAA GCA GCG TAT CC 3’. The cycle used in a Bio-Rad S1000 Thermal Cycler for amplification was as follows, 1. 95°C for 15 minutes for initial activation 2. 94°C for 1 minute for denaturing 3. 53°C for 30 seconds for annealing 4. 72°C for 1 minute for extension 5. Steps 2-4 repeated 30 times 6. 72°C for 10 minutes for final extension 7. 4°C forever reaction completed. PCR product was visualized with 10× loading buffer: 30% glycerol (EMD Millipore GX0185-2), 0.2% Orange G (Eastman Organic Chemicals 16230), and 69.8% ddH₂O. One microliter of product was placed into wells of a 2% by weight agarose gel (Sigma A9539-250g) containing 52 wells and Gel Star nucleic acid stain (Lonza Cat No.50535). Samples were run at 80V and imaged on a Bio-Rad ChemiDoc XRS+ System. Sample bands were compared to DNA ladder (Lonza Cat No.50461), positive gDNA, negative gDNA, and negative H₂O control bands; Cre is ~350bp, wildtype tdTomato is ~297bp and mutant tdTomato is ~196bp. Mice were maintained at homozygosity at both alleles.

Alcohol Exposure

Homozygous Nestin-CreER^T2: tdTomato female mice were bred with homozygous male mice of the same strain. At determination of pregnancy by weight gain, females were single housed in new cages containing nesting materials (shredded paper, kimwipes and a cardboard house) separate from the male breeder. Once offspring were born, each cage containing mother and litter was randomly assigned to either EtOH or air (control) groups. Cages containing mothers and pups were placed into vapor inhalation chambers from PND 3 through PND 15. Either EtOH vapor or air (control) was pumped into the chamber for 4 hr per day between 10:00-14:00 hr, as previously described (Newville et al., 2017). EtOH vapor levels were measured at the end of each 4 hr exposure period using a breathalyzer (Intoximeters, St. Louis, MO, 12-0050-00). Fresh chow was provided after exposure to avoid continued EtOH exposure via consumption of EtOH-contaminated pellets. EtOH vapor concentrations were gradually increased from PND 3 to PND 9 allowing acclimation, and then held steady for the remaining days PND 10 to PND 15 (7.82 ± 0.7g/dl, n=4). We previously determined that this paradigm produces the following BECs: PND 4 (128.2 mg/dl), PND 7 (160.0 mg/dl), PND 11 (185.6 mg/dl), PND 14 (167.7 mg/dl) (Newville et al., 2017) (Figure 1). This exposure paradigm results in an overall peak at postnatal day 12 and a daily peak exposure at 4 hours, coinciding with the end of the exposure. The blood alcohol levels diminish to 0 mg/dl approximately 12 hours following the start of exposure (Morton et al., 2014).

Figure 1.

Alcohol Vapor Concentration. Comparison of vapor ethanol concentrations achieved in the third-trimester equivalent exposure paradigm used by Newville et al., 2017 in a previous study (purple dashed line) and our current study (orange solid line). The dashed vertical line represents peak alcohol exposure on postnatal day 12.Two-way ANOVA determined no significant difference across studies (F (1, 13) =2.423 P=0.1436). Pup blood ethanol concentrations (BECs) were taken at 4 time points in the previous study.

Assessment of dDGCs

To assess the impact of EtOH exposure on the production and/or long-term survival of dDGCs born during the early postnatal period, all pups (n) received a single injection of tamoxifen (33 mg/kg) at PND 2, 24 hr prior to the first EtOH exposure on PND 3. Male mice from each litter (L) were sacrificed on PND 16 (24 hr following the final vapor chamber exposure; EtOH n=4 L=4, Air n=6 L=6). The remaining male mice were reared under standard housing conditions until sacrifice at PND 50 (EtOH n=5 L=4, Air n=6 L=4).

Assessment of aDGCs

To assess the effects of early postnatal EtOH exposure on adult neurogenesis, mice were segregated by sex at weaning and reared in SH or EE conditions. SH conditions included a standard mouse cage (28cm × 18cm × 13 cm) and a cardboard house with 2-5 mice per cage. EE conditions included a larger cage (48cm × 27cm × 20 cm) with 2 running wheels and multiple objects (ladder, tunnel, hanging toys and plastic house) changed weekly with 4-7 mice per cage (Kajimoto et al., 2013). At PND 35-49, each mouse received a daily single injection of tamoxifen (180 mg/kg, i.p.) for 5 consecutive days. Mice were sacrificed at PND 78-92 (i.e., 6 weeks post-tamoxifen: EtOH-EE, n=11 L=3; Air-EE n=5 L=3; EtOH-SH n=8 L=3; Air-SH n=8 L=4). Analysis was restricted to male offspring due to the limited availability of mice for these studies; future studies will characterize the effects on female offspring, as previous work using rodent models have established sex differences in vulnerability to alcohol toxicity (Sharrett-Field et al., 2013, Goodlett and Peterson, 1995).

Tamoxifen Administration

Tamoxifen (T5648; Sigma-Aldrich, St. Louis) was dissolved in 10% EtOH (made from 200 proof; 459836; Sigma-Aldrich)/90%sunflower seed oil (S5007; Sigma-Aldrich). Tamoxifen solution was administered intraperitoneally (IP) to pups at PND 2 (33 mg/kg) or young adult mice (180 mg/day) using the dosing schedule described above. Previous work by Lagace et al., 2007 demonstrate that the tamoxifen injection paradigm described above elicits maximal recombination in approximately 97% of stem-like cells in the SGZ of NestinCreER^T2/R26R-YFP mice. We have not observed labeling of hippocampal progenitors in NestinCreER^T2:tdTomato mice in the absence of tamoxifen.

Immunohistochemistry

All mice were sacrificed by overdose with sodium pentobarbital (Fort Dodge Animal Health, Fort Dodge, IA, NDC 0856-0471-01) and immediately transcardially perfused with phosphate buffered saline (PBS) containing 0.1% procaine hydrochloride (Sigma-Aldrich P9879) and 2U/mL heparin (Sigma-Aldrich H3393), followed by 4% paraformaldehyde (Sigma-Aldrich P6148) in 0.1M PBS. Brains were post fixed overnight and stored in a 30% sucrose solution in 0.1M PBS at 4°C. Cerebral hemispheres were sectioned at 30 μm thickness in the coronal plane using a freezing sliding knife microtome (Phoenix Equipment Inc., Rochester, NY, 14621) and stored at −80°C in cryoprotective solution. Floating sections were processed for immunofluorescence as previously published by (Li et al., 2010a). Sections containing dorsal hippocampus were immunolabeled for NeuN, a neuron specific nuclear protein, using mouse anti-NeuN primary antibody (1:1000; EMD Millipore MAB377) and FITC-conjugated donkey anti-mouse secondary antibody (1:250; Jackson Immunoresearch Laboratories, West Grove, PA). Tissue was counterstained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher Scientific, Carlsbad, CA D1306) and coverslipped using Fluoromount G mounting medium (Electron Microscopy Sciences, Hatfield, PA, 17984). Z-stack maximum projection images were obtained using LASX acquisition software with a Leica DMi8 TCS SP8 confocal microscope (Wetzlar, Germany). A 20x oil immersion objective was used to obtain images that were subsequently tiled and stitched using LASX or Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA).

Stereology

The number of tdTom⁺/NeuN⁺ co-labelled neurons were counted within serial coronal sections using the Optical Fractionator probe in Stereoinvestigator software (Microbrightfield Bioscience, Williston, VT) linked to an Olympus IX-81 DSU spinning disk confocal microscope with a 40x objective (Shinjuku, Tokyo, Japan) as previously described (Gustus et al., 2018). A region of interest (ROI) contour was manually outlined within each section using a 10× objective. ROI contours followed the outer limits of the granule cell layer and included one cell thickness into the hilar region. ROIs at the level of dorsal hippocampus, determined by the presence of the medial habenula, were analyzed between anterior-posterior coordinates −1.54 to −2.30 mm relative to bregma. The optical dissector height was set to 15 μm, with top and bottom guard zone heights at 2 μm each. For estimating the number of tdTom⁺ dDGCs at PND 16 and PND 50, the counting frame size was 25 μm² within a 60 μm² sampling grid, resulting in ten or more sampling sites per histological section and 2-5 cells per counting frame. For estimating the number of tdTom⁺ aDGCs, we utilized an exhaustive counting scheme, in which the total number of tdTom⁺/NeuN⁺ cells within each ROI was counted. We sampled every 8^th section, resulting in stereological sampling across 3 out of 24 histological sections per mouse for each experiment. ROI volumes were consistent across treatment groups at each respective time point (F (1, 17) = 0.002292 p= 0.9624). All data are expressed as the mean number of tdTom⁺/NeuN⁺ cells/mm³ /mouse ± S.E.M., which was calculated by dividing cell number per histological section by volume of the corresponding contours for each histological section, averaged across 3 histological sections per mouse. All stereological counts were performed by investigators blinded to treatment.

Statistical Analysis

Data were subjected to two-way ANOVA using GraphPad Prism 8.0 (La Jolla, CA). Data are expressed as means ± SEM, with p<.05 considered statistically significant. The statistical unit of determination (n) was defined as one mouse, sampled across 3-6 litters for each experiment to avoid litter effects (Mirescu et al., 2004, Loi et al., 2014).

Results

Impact of early postnatal EtOH vapor exposure on the generation and long-term survival of dDGCs

We first assessed the impact of early postnatal EtOH exposure on the production and long-term survival of dDGCs generated during the period of exposure. For these experiments, dDGCs were labeled by administration of single dose of tamoxifen at PND 2. Pups were then exposed to EtOH vapor or air (control) vapors for 4 hr/day from PND 3 through PND 15 and sacrificed at PND 16 or PND 50 (Figure 2A). As shown in Figures 2B and 2C, a single dose of tamoxifen at PND 2 resulted in robust tdTom expression in a cohort of dDGCs at both PND 16 and PND 50. tdTom⁺ dDGCs were highly concentrated in the inner 2/3 region of the dentate granule cell layer, in agreement with previous reports regarding the distinct layering pattern of dDGCs (Mathews et al., 2010). A single dose of tamoxifen at PND 2 resulted in tdTom labeling of approximately 350,000 dDGCs/mm³ at PND 16. This cohort of tdTom⁺ dDGCs was surprisingly stable in number out to PND 50, with no main effect of age (F (1, 17) = 0.08, p = 0.775; Figure 2D). The mean number of tdTom⁺ dDGCs was decreased by 17-19% in EtOH-treated groups, although this did not reach statistical significance (F (1, 17) = 1.765, p = 0.20), and there was no significant interaction (F (1,14) = 0.006, p = 0.93).

Figure 2.

Effect of early postnatal EtOH vapor exposure on production and survival of dDGCs. (A) Experimental timeline. Tamoxifen (TAM), Sacrifice (Sac) (B) Triple fluorescence confocal images through the dentate gyrus of dorsal hippocampus from tamoxifen-treated P16 or P50 nestin-CreER² :tdTomato mice exposed to air or EtOH vapors from P3 through P15. tdTom⁺ dDGCs (red) were co-labeled for (NeuN (green) and counterstained with DAPI (blue). (C) High power (63×) representative confocal images of the granule cell layer in PD 16 (left) and PD 50 (right) dorsal hippocampus, demonstrating co-localization of tdTomato and NeuN. (D) Quantification of co-labeled NeuN⁺/tdTom⁺ DGCs; n=mouse, L=litter.

Early postnatal alcohol exposure does not alter the production of aDGCs or the EE-mediated neurogenic response

We previously demonstrated that adult mice exposed to moderate levels of EtOH throughout gestation display marked impairment in their ability to mount a neurogenic response to EE, with no change in the size of the hippocampal progenitor pool (Choi et al., 2005, Kajimoto et al., 2013). To determine whether a similar neurogenic deficit occurs in mice exposed to EtOH during the early postnatal period, pups were exposed to EtOH vapors or air as in the previous experiment, but were placed into either SH or EE conditions at one week post-weaning. For this experiment, aDGCs were genetically labeled by tamoxifen administration once daily for 5 consecutive days at PND 35-39 of age and sacrificed at approximately 6 weeks post-tamoxifen (PND 78-92; Figure 3A).

Figure 3.

Effect of early postnatal EtOH vapor exposure on the production of aDGCs and the neurogenic response to EE. (A) Experimental timeline. Tamoxifen (TAM), Sacrifice (Sac) (B) Dual fluorescence confocal images through the dentate gyrus of dorsal hippocampus from tamoxifen-treated nestin-CreER ² :tdTomato mice housed in enriched (EE) or standard (Std) conditions, exposed to air or EtOH vapors from P3 through P15. tdTom⁺ aDGCs (red) co-labeled for NeuN (green). (C) Representative confocal images of aDGCs in enriched (left) and standard (right) animals, co-expressing tdTomato and NeuN. (D) Quantification of tdTom⁺ aDGCs.

As shown in Figures 3B and 3C, this dose of tamoxifen labeled a cohort of tdTom⁺ aDGCs localized primarily to the innermost 1/3 region of the dentate granule cell layer across all groups. The multi-day tamoxifen dosing regimen in young adulthood (PND 35-39) labeled approximately 17.5-fold fewer tdTom⁺ aDGCs under SH compared to the number of dDGCs labeled by PND 2 tamoxifen. As anticipated, rearing mice in EE resulted in a robust, 2.5-fold increase in the number of tdTom⁺ aDGCs. This neurogenic response occurred regardless of EtOH treatment. Although there was a main effect of housing (F (1, 28) = 15.4, p =0.0005), there were no main effects of EtOH treatment (F (1, 28) =0.05, p=0.81) or housing × treatment Interaction [F (1, 28) =0.02, p=0.88)] (Figure 3D). These observations indicate that early postnatal EtOH exposure has no impact on the production and/or survival of aDGCs or on the neurogenic response to EE in adulthood.

Discussion

In the present study, we utilized NestinCreER^T2:tdTomato bitransgenic mice to elucidate the impact of EtOH exposure on hippocampal dentate neurogenesis during the early postnatal period and in adulthood. Using this labeling approach, we found that EtOH exposure had no statistically significant effect on the production or the survival of tdTom⁺ dDGCs when EtOH was administered by vapor chamber from PND 3-PND 15. Further, we found no effect of early postnatal EtOH exposure on the production of aDGCs or on the neurogenic response to EE in adulthood. Collectively, these results suggest that neither early postnatal nor adult dentate neurogenesis is significantly influenced by moderate levels of EtOH administered by vapor chamber during the early postnatal period in mice.

We found that a single injection of tamoxifen to Nestin-CreER^T2:tdTomato mice at PND 2 robustly labeled a cohort of dDGCs in all mice. The labeled dDGCs were essentially stable, with no significant change in cell number out to PND 50, regardless of EtOH exposure. Although there was no statistical main effect of EtOH on the number of tdTom⁺ dDGCs at either PND 16 or PND 50, a statistically non-significant reduction in the mean number of tdTom⁺ dDGCs in EtOH-exposed mice at both ages is noteworthy. Based on prior stereological estimates of total DGC number at PND 10 (Bonthius et al., 2004), we estimate that a single dose of tamoxifen at PND 2 results in labeling of ~24% of the total dDGCs population. Early studies utilizing Nissl-stained histological sections to estimate total DGC number demonstrated a significant reduction (~10-11%) in total DGCs (assessed at PND 10 or PND 35) following high dose EtOH (BECs ~340-360 mg/dl), delivered via gavage during the peak early postnatal DGC proliferative phase (Livy et al., 2003, Miller, 1995, West et al., 1986, Gil-Mohapel et al., 2010). Our results also suggest that a more moderate dose of EtOH delivered by a less strenuous inhalation method may also impair the production of dDGCs, although this effect did not reach statistical significance in our study. In addition to differences in routes of administration, which can affect the dynamics of BEC levels, sampling differences and differences in peak ethanol concentrations may explain discrepancies between studies. For example, we sampled only a single cohort of tdTom⁺ dDGCs and limited our sampling to the dorsal hippocampus. Bird et al.(Bird et al., 2019), recently described a significant ~20% decrease in the mean number of neuroblasts within ventral but not dorsal hippocampal dentate of adult mice when exposure to daily EtOH vapors spanned from mid-gestation into early postnatal life, although the impact on dDGCs was not determined. We previously demonstrated that the EtOH exposure paradigm and dosing used in the current study results in acute impairment of oligodendrocyte proliferation and persistent white matter injury in Nestin-CreER^T2:tdTomato mice, as assessed by histological, biochemical and diffusion-tensor MRI indices (Newville et al., 2017). More recent studies have also demonstrated both acute cell death and long-term loss of GABAergic interneurons following daily EtOH exposure via vapor chambers from PND 2 - PND 9 (average BECs ~220 mg/dl)(Bird et al., 2018). Thus, our current findings indicate a relative resistance of early postnatal neurogenesis to levels of EtOH that disrupt other aspects of postnatal brain development. It is likely that a higher EtOH dose, which is more difficult to achieve using vapor inhalation vs. gavage delivery (Morton et al., 2014), and/or more prolonged EtOH vapor exposure beginning in the gestational period, is necessary to achieve significant loss in the production and or survival of dDGCs.

Administration of tamoxifen daily for five days during young adulthood (PND 35-PND 39) labeled a cohort of tdTom⁺ aDGCs that was situated primarily within the inner third of the dentate granule cell layer, as previously described by us and others (Cunningham et al., 2017, Kajimoto et al., 2013, Lagace et al., 2007, Mathews et al., 2010). Prior studies with rats have demonstrated a persistent deficit in adult neurogenesis following early postnatal EtOH binge-like exposures using intragastric gavage between PND 4-10 (BACs ~315 mg%), which could be reversed by a sequential wheel-running and enriched environment regimen (Hamilton et al., 2011, Klintsova et al., 2007, Hamilton et al., 2012). Importantly, we found that early postnatal EtOH exposure also had no impact on the neurogenic response to EE. This result is surprising, since we have previously demonstrated a robust impairment of EE-mediated neurogenesis in adult mice exposed to moderate doses (80-120 mg/dl) of EtOH throughout gestation (Choi et al., 2005, Cunningham et al., 2017, Kajimoto et al.,2013). The impaired EE-mediated neurogenesis in gestationally exposed mice is also associated with a significant impairment of pattern discrimination learning (Kajimoto et al., 2013) and with a compensatory increase in the frequency of excitatory synaptic activity in surviving aDGCs (Kajimoto et al., 2016). Gil-Mohapel et al. (Gil-Mohapel et al., 2014) also demonstrated significant effects of gestational EtOH exposure (maternal BEC 143.31 +5.31) on the production of aDGCs, during senescence (>1 year old mice), but not in adolescence/young adulthood. It is interesting that the persistent impairment of EE-mediated adult dentate neurogenesis by lower doses of EtOH in utero are not replicated by our early postnatal exposure paradigm. This suggests that gestational exposures must exert a unique impact on long term brain development and plasticity. The basis of this is currently unclear, but could potentially be explained by critical periods for epigenetic changes to circuit function during embryonic development and/or additional/indirect factors from the fetal-placental unit, which are absent in the postnatal exposure paradigm Interestingly, baseline adult neurogenesis under standard housing has previously been shown to be impaired following higher dose postnatal alcohol exposures, an effect that can be rescued by voluntary exercise and/or enriched environments (Boehme et al., 2011, Christie et al., 2005, Redila et al., 2006).

Consuming alcohol during the 3rd trimester of pregnancy (weeks 28-40 in humans; postnatal day PND 1-10 in rodents), when the production of dentate granule cells is at its peak, is not uncommon (Dobbing and Sands, 1979, Yamamoto et al., 2008, Tamaki et al., 2008, Leonardson et al., 2007). Unlike the mouse where hippocampal development occurs at a rapid rate during the first 2 weeks of life, human hippocampal development occurs mostly in utero during the second and third trimester. The current study suggests that a relatively moderate level of EtOH exposure limited to the early postnatal period in mouse (3^rd trimester equivalent for human brain growth spurt) does not impair the production or survival of either dDGCs or aDGCs, and has no effect on the EE-mediated neurogenic response in adulthood. Future studies will be needed to determine whether the function or connectivity of postnatally-generated DGCs is altered by this exposure paradigm.