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. Author manuscript; available in PMC: 2021 May 21.
Published in final edited form as: Neuroscience. 2020 Apr 3;435:124–134. doi: 10.1016/j.neuroscience.2020.03.046

Single-day postnatal alcohol exposure induces apoptotic cell death and causes long-term neuron loss in rodent thalamic nucleus reuniens.

Zachary H Gursky a, Emma C Spillman a, Anna Y Klintsova a
PMCID: PMC7236664  NIHMSID: NIHMS1581814  PMID: 32251710

Abstract

Fetal alcohol spectrum disorders (FASD) constitute a prevalent, yet preventable, developmental disorder worldwide. While a wealth of research demonstrates that altered function of hippocampus and prefrontal cortex may underlie behavioral impairments in FASD, only one published paper to date has examined the impact of developmental alcohol exposure on the region responsible for coordinated prefrontal-hippocampal activity: thalamic nucleus reuniens (Re). In the current study, we used a rodent model of human third trimester alcohol exposure to examine both the acute and lasting impact of a single-day alcohol exposure on Re. We administered 5.25 g/kg of ethanol to male and female Long Evans rat pups on postnatal day (PD) 7. We used unbiased stereological estimation to evaluate cell death or cell loss at three time points: 12 hours after alcohol administration; 4 days after alcohol administration (i.e., PD11); in adulthood (i.e.,postnatal day 72). Alcohol exposure on PD7 increased apoptotic cell death in Re on PD7, and caused short-term cell loss on PD11. This relationship between short-term cell death versus cell number suggests that alcohol-related cell loss is driven by induction of apoptosis. In adulthood, alcohol-exposed animals displayed permanent cell loss (mediating volume loss in the Re), which included a reduction in neuron number (relative to procedural controls). Both procedural controls and alcohol exposed animals displayed a deficit in non-neuronal cell number relative to typically-developing controls, suggesting that Re cell populations may be vulnerable to early life stress as well as alcohol exposure in an insult- and cell type-dependent manner.

Keywords: Stereology, Histology, Neuroanatomy, Fetal Alcohol Spectrum Disorders, Immunocytochemistry

Introduction

Estimated prevalence of fetal alcohol spectrum disorders (FASD) is between 1-5% in the United States (May, Chambers et al. 2018), and may be higher in other countries (Roozen, Peters et al. 2016). FASD is an umbrella term that covers a wide variety of alcohol-related disorders and often manifest as deficits in cognition, executive function, learning and memory (Hoyme, Kalberg et al. 2016). Underlying behavioral outcomes of FASD, alcohol-induced damage to the brain occurs in a dose- and timing-dependent manner, particularly during periods of synaptogenesis in a given region (Ikonomidou, Bittigau et al. 2000).

A large body of literature has demonstrated that damage to hippocampus (HPC) structure and function is present in many manifestations of FASD (reviewed in Berman and Hannigan 2000). Deficits in complex behaviors (collectively, “executive function” (EF)) are also prevalent in FASD (Khoury, Milligan et al. 2015). Khoury, Milligan et al. (2015) determined that working memory, inhibitory control, and set shifting (all of which fall under EF) are impaired in FASD. Mechanistic work using rodents to examine the neuroanatomical underpinnings of EF domains indicate that the HPC and medial prefrontal cortex (mPFC) must remain intact and functionally coordinated to facilitate behaviors such as working memory (reviewed in Bissonette, Powell et al. 2013, demonstrated in Hallock, Wang et al. 2016). Even traditionally HPC-dependent tasks which are negatively affected by third trimester-equivalent alcohol exposure (AE) in rodents, such as context pre-exposure facilitated fear conditioning (Murawski, Klintsova et al. 2012), require both HPC and mPFC (Heroux, Robinson-Drummer et al. 2017).

While HPC sends direct monosynaptic projections to mPFC in both primate and rodent brain (Roberts, Tomic et al. 2007, Varela, Kumar et al. 2014), the most prominent and direct pathway for information flow from mPFC to HPC is through thalamic nucleus reuniens (Re). Re, a ventral midline thalamic nucleus found in both primates and rodents, is reciprocally connected to both mPFC and HPC (Varela, Kumar et al. 2014). Inactivation or lesion of Re has been demonstrated to impair mPFC-HPC communication (Hallock, Wang et al. 2016, Roy, Svensson et al. 2017), and cause behavioral deficits in EF (Hallock, Wang et al. 2016, Linley, Gallo et al. 2016). Due to the critical role of mPFC-Re-HPC circuitry in EF, the prominence of EF impairment in FASD, and the necessity Re for coherent mPFC-HPC activity, it is likely that the circuit comprised of mPFC-Re-HPC undergoes structural and functional impairment following developmental alcohol exposure.

While previous studies have shown significant vulnerability of both prefrontal cortex and thalamus to apoptotic cell death following single-day developmental alcohol exposure in rat (Ikonomidou, Bittigau et al. 2000), little attention has been given to the functionally distinct subdivisions within each structure, as well as the long-term impact of such cell death. Re, the critical intermediary for mPFC-HPC coordination, is almost entirely unexamined in the context of FASD. The first and only study to examine the Re following developmental alcohol exposure, Gursky, Savage et al. (2019), observed robust neuron loss, and volume loss in Re of adult female rats exposed to alcohol on PD4–9. Alcohol exposure did not appear to influence the immediately adjacent thalamic rhomboid nucleus. While this suggests that third trimester-equivalent alcohol exposure impacts ventral midline thalamus in a nucleus-specific manner, it was limited in its investigation of a single sex (i.e., females). Regardless, we believe the specificity of damage observed is linked to the central role of Re in mPFC-Re-HPC circuitry, and is likely to occur in both sexes.

The current study substantially extends the hypothesis that Re is particularly vulnerable to third trimester-equivalent alcohol exposure, first demonstrated in Gursky, Savage et al. (2019), by examining the impact of single-day postnatal alcohol exposure on the integrity of prefrontal-thalamic circuitry throughout life. By examining three timepoints in Re development (two early postnatal timepoints and adulthood, presented in timeline format in Figure 1), we test the hypothesis that the mechanism by which third-trimester binge alcohol exposure causes long-term neuron loss in Re is through acute apoptosis-induced cell deficit in both male and female rats. This hypothesis directly opposes the possibility that a deficit in neuron number arises during adolescence or adulthood due to altered neuronal survival throughout life.

Figure 1: Experimental timeline of all animals and tissue generated.

Figure 1:

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Rat pups for all time points were injected with trace amounts of black India ink for identification on postnatal day (PD) 3. Alcohol exposed pups (AE) and sham intubated (SI) control pups had blood drawn on PD7, 90 minutes after the second dose of ethanol. Suckle control (SC) pups were weighed on PD7, then left undisturbed until either tissue extraction (PD7 and PD11) or weaning (PD72). All pups for sacrifice at PD72 were weaned on PD23.

We utilize linear mixed effects modeling to maximize power and generalizability of our observations while accounting for within-animal or within-litter clustering effects (Galbraith, Daniel et al. 2010). We also examine whether the influence of alcohol on Re volume (previously identified by Gursky, Savage et al. (2019)) is mediated by loss of cells (either in total, or of specific phenotype) using mediation modeling and bootstrapping to increase robustness given smaller sample sizes commonly observed in experimental biology research. Together, these analytical approaches allowed us to demonstrate the susceptibility of Re to single-day developmental alcohol exposure, and the likely apoptotic mechanism by which long-term neuron loss occurs in Re.

Experimental Procedures

Experimental Subjects

All procedures were carried out in accordance with the animal use protocol approved by University of Delaware Institutional Animal Care and Use Committee and in accordance with NIH’s Animal Care Guidelines. An experimental timeline is provided in Figure 1. Experimental animals were bred in-house at the University of Delaware by housing female Long-Evans rats with experienced male breeders. Breeding animals were acquired from Envigo (Indianapolis, IN) and housed in cages of standard dimensions (17 cm high x 145 cm long x 24 cm wide) in a 12/12 hr light cycle upon arrival. All dates are derived relative to gestational age (i.e., the discovery of a seminal plus was considered gestational day 0; gestational day 22 was considered “postnatal day 0”). On PD3, each litter was culled to 10 pups (5 male and 5 female) when possible, and a small amount of non-toxic India black ink into the paws for identification. Litters were then assigned to be either SC, or experimental (split-litter design containing the same number of both SI controls and AE pups). 89 total animals (n = 5 per sex per treatment per experiment, except PD11 AE males in Experiment 3, where n = 4) from 10 litters (NExperiment 1=3, NExperiment 2=4, NExperiment 3=3) were generated for this study. On PD7, all AE pups received 5.25 g/kg/day alcohol in a milk formula in 2 doses, 2 hours apart via intragastric intubation. Milk and milk/ethanol treatment formulas were prepared from a base milk formula prepared according to the previously described method and were delivered via intragastric intubation (e.g., Helfer et al., 2009). On PD7 pups of the AE group were given 2 intubations of the milk / ethanol formula containing 11.9% (v/v) ethanol, with a 2-hour interval between intubations. Each intubation delivered 2.625 g/kg of ethanol resulting in a total daily dose of 5.25 g/kg for each animal. For each round of intubations, the pups were removed from the dam as a litter and kept on a 37°C heating pad. A premeasured length of PD-10 tubing was lubricated with corn oil and gently inserted down the pup’s esophagus into the stomach and the milk/ethanol formula was infused over a 20-second period. SI pups were intubated alongside the AE pups but received no liquid solution, to control for the stresses of intubation and maternal separation. AE pups also received 2 supplementary feedings of milk at 2 and 4 hours following the second alcohol dose on PD7 to reduce the confound of nutritional deficits following AE, as is common in rodent studies of early postnatal alcohol exposure (Helfer, Calizo et al. 2009, Monk, Leslie et al. 2012). SC pups remained undisturbed except for daily weighing.

Blood Alcohol Content

Blood samples were collected from AE animals 90 minutes after the second alcohol exposure, when peak blood alcohol content (BAC) is observed (Kelly, Bonthius et al. 1987). Blood samples (10 ul) were collected from the tip of the tail clip of each intubated pup into heparinized 20-ul capillary tubes. Blood samples were centrifuged at 15,000 x g for 25 mins, and plasma was collected and stored at −20°C until analysis. SI animals had blood samples collected to account for procedural stress, but these samples were not analyzed. BAC was analyzed using an Analox GL5 Alcohol Analyzer (Analox Instruments, Boston, MA).

Weaning

Animals for PD72 (Experiment 2) were weaned on PD23; animals were subsequently housed in cages of 3 same-sex animals, as is standard among experiments utilizing rodent models of development alcohol exposure.

Tissue Preparation

Brain tissue was collected at one of the following time points: PD7 (12 hrs after initial AE), PD11, or PD72. Animals were deeply anesthetized using ketamine and xylazine, and transcardially perfused using 0.1M phosphate buffered saline (PBS; pH = 7.20) followed by 4% paraformaldehyde (in PBS; pH = 7.20). Brains were postfixed in 4% paraformaldehyde for 48 hours, and equilibrated in 30% sucrose in 4% paraformaldehyde 3 times prior to sectioning. Forebrain was exhaustively sectioned in coronal plane at 40μm and collected in cryoprotectant solution.

Cresyl Violet

Every 6th section of forebrain was systematically slide-mounted and dried for 24-48 hours at room temperature. Tissue was then stained with Cresyl violet (Electron Microscopy Sciences, Hatfield, PA, USA, Catalog # 12780), and cover slipped with DPX mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA, Catalog # 13512). All 89 animals (n = 4-5 per sex per treatment per age at sacrifice) were prepared for neuroanatomical cresyl violet analysis. However, some tissue could not be analyzed due to sections’ fragility during tissue processing (final N’s = 3-5/sex/treatment/age at sacrifice, see Supplemental Material, Table 2). Apoptotic cells were easily identified in sections containing Re 12 hours after AE. Apoptotic cells were defined as such by the presence of at least 1 densely stained spherical particle, an apoptotic body (labeled with * in Fig. 2 B & C). Where multiple small apoptotic bodies occurred within an area less than that of a nonapoptotic cell, they were classified as representing a single apoptotic cell (see figure 2C for examples).

Figure 2: Alcohol exposure (AE) on postnatal day 7 increases apoptotic cell death in thalamic nucleus reuniens.

Figure 2:

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A representative outline of thalamic nucleus reuniens (Re) can be found in panel 2A (3V indicates the third ventricle; scale bar = 1mm). Representative photomicrographs from a sham intubated (SI) and an alcohol-exposed (AE) animal (panels 2B and 2C, respectively) demonstrate representative quantification of apoptotic cells based on clustering of apoptotic bodies within tissue (scale bars = 10 μm). Each dotted contour would have been identified as 1 apoptotic cell by the experimenters Only AE females (solid bars) had altered volume of Re relative to any other group (panel 2D; males represented by striped bars). SI did not alter apoptotic cell death, while AE increased apoptosis approximately 30-fold (panel 2E). All data in this figure are separated by sex due to the sex-by-treatment interaction observed in panel 2E. Graphs present raw data points (in grey) with black bars representing mean ± SEM for that group. Groups with similar letters over them indicate that those groups did not significantly differ.

NeuN Immunocytochemistry

For quantification of Neurons in Re at PD72, we performed immunocytochemistry for the neuronal marker NeuN. For cell type analyses in Experiment 2, all available remaining tissue (subset of 6 animals per postnatal treatment) was used. Every 6th section between anterior-posterior coordinates −0.8mm and −4.0mm relative to bregma were selected for free-floating immunocytochemistry. Tissue was first washed in deionized H2O followed by 0.1M tris buffered saline (TBS; pH = 7.40). After washing, tissue was exposed to 0.6% peroxide in TBS to quench endogenous peroxidases. Tissue was then washed in TBS again before a 1 hour incubation in a TBS-based blocking solution, containing 1% Triton X-100 (Millipore Sigma, product # X100), 3% normal goat serum (Millipore Sigma, product # S26) in TBS, to eliminate non-specific antibody binding. Immediately following blocking, tissue was transferred to primary antibody (anti-NeuN made in mouse, Millipore, product # MAB377) diluted 1:500 in blocking solution, and incubated at 4°C for 48 hours. Negative control sections were incubated in blocking solution with no antibody added; no visible labeling was present in negative controls. After primary antibody, tissue was washed in TBS, then incubated in secondary antibody (anti-mouse made in goat, Vector Labs, product # BA-9200) diluted 1:200 in blocking solution at room temperature for 1 hour. Both positive and negative control sections were incubated in secondary antibody. Tissue was subsequently washed and incubated in ABC solution (Vector labs, product # PK-6100) at room temperature for 1 hour. After washing in TBS, tissue was incubated in Nickle-enhanced 3,3′-Diaminobenzidine (DAB), activated with peroxide, at room temperature for approximately 4:00 minutes. Tissue was then washed and slide mounted in TBS, dried at 50°C using a slide warmer for approximately 4 hours, counterstained using Pyronin Y (Electron Microscopy Sciences, product # 19560), and coverslipped using DPX mounting medium.

Unbiased Stereological Estimation

The optical fractionator probe (Stereo Investigator, MBF Bioscience, Williston, VT) was used to estimate the number of cells containing apoptotic bodies (PD7), total cell number (PD11 and PD72), or total neuron number in Re. The entire rostrocaudal extent of Re was counted. Regions were outlined using a 5x objective on a Zeiss Axioskop 2 Plus (Carl Zeiss Inc., Thornwood, NY).

Regions for PD72 time point were outlined consistent with the following regions in Paxinos and Watson (2013). Reuniens (“Re”). Due to the early developmental stage of the PD7 and 11 brains relative to the adult rat briain, we used a developmental rat brain atlas to define the Re (Paxinos, Ashwell et al. 1994). The following anatomical landmarks were used to delineate the contour of reuniens at several points throughout its rostral-caudal extent:

At approximately 1.08mm caudal to bregma in the rat brain, the medial border of Re is against the high-density band of cells identified as the paraventricular thalamic nucleus. The lateral border wraps around the bed nucleus of the stria terminalis, with the ventral tip adjacent to the fornix. The ventral border rests against the paraventricular nucleus of the hypothalamus. The dorsal border follows the anteromedial thalamic nucleus.

At approximately 2.04mm caudal to bregma, the ventral border of Re extends along the paraxiphoid nucleus of thalamus, ranging from fairly level to largely indented in the middle. The lateral borders are visible as the boundary between the roughly triangular ventral reuniens nuclei and the submedium thalamic nuclei. The dorsal border runs along the more cell-dense rhomboid nucleus of thalamus.

At approximately 2.52mm caudal to bregma, the ventral border runs along the paraxiphoid nucleus of the thalamus, with a small indent where the xiphoid thalamic nucleus impinges on Re. The lateral border runs along the ventral portion of the submedius nucleus of thalamus, and did not include the ventral reuniens thalamic nucleus. The dorsal border extended until Re meets the more cell-dense rhomboid nucleus.

At approximately 3.36mm caudal to bregma, the dorsal border of Re runs along the central medial thalamic nucleus, and extends laterally until the superior cerebellar peduncles. The lateral and ventral borders are a visible arc shape constrained between the superior cerebellar peduncles (laterally) and the prosomere 3 (which indents the ventral tip of Re, slightly).

Quantification of apoptotic cell number, total cell number, and total neuron number were performed using a 40x objective. For cresyl violet stained brains (i.e., to apoptotic cell number and total cell number), a dissector height of 12 μm and a guard zone of 2 μm was used. The sampling grid was set to 200 × 200 μm and the counting frame set to 40 × 40 μm. For neuron quantification, a dissector height of 15 μm and a guard zone of 2 μm was used. The sampling grid was set to 200 × 200 μm and the counting frame was set to 50 × 50 μm. The mean coefficients of error (Gundersen, m=1) for all estimates can be observed in Supplemental Material Table 2.

Our stereological estimates maintained high degrees of reliability due to low coefficients of error (CEs), averaging around or below 0.1, a recognized indicator of appropriately sampled and reliable stereological estimation (Slomianka and West 2005). In instances where CE values for stereological estimation was drastically higher (i.e., 0.2 or above), such was only the case in estimating the relatively low amounts of naturally-occuring apoptosis in control groups (both SI and SC), as has been observed by others examining alcohol-induced cell death (Smith, Guevremont et al. 2015). The necessary sampling required to reduce CE values to moderate levels would have resulted in vast over-sampling in AE brains. Nevertheless, we must keep this in mind when interpreting the exact values of the stereological estimates of apoptotic cell number in SC and SI, regardless of the clear and obvious between-treatment impace of alcohol exposure on apoptotic cell number in these regions.

Statistical Analysis

All analyses were performed using RStudio version 1.1.414 (RStudio Team 2016) running the “tidyverse” (Wickham 2017), “mediation” (Tingley, Yamamoto et al. 2014), “Ime4” (Bates, Mächler et al. 2015), and “ImerTest” (Kuznetsova, Brockhoff et al. 2017) packages, α = .050 was used for all analyses.

Linear mixed-effects models (LMM) were generated for each analysis using the “Ime4” package. Degrees of freedom, t-values, and p-values were generated using the ImerTest package, implementing the Satterthwaite’s method (Giesbrecht and Burns 1985). The use of LMM allows clustering variables to be appropriately accounted for as random effects in designs that examine non-independent data points, such as analysis of multiple animals per litter or multiple measures within the same animal (Galbraith, Daniel et al. 2010). Results from LMM analysis are presented as the regression coefficient (β), plus or minus standard error, followed by the df as estimated by Satterthwaite’s method, t-value, and p-value.

Mediation analysis (using the “mediation” package) of the influence of alcohol on reuniens volume used 2 mixed effects models (from the “Ime4” package). The outcome model was volume regressed on postnatal treatment (fixed effect), sex (fixed effect), total cell/neuron/non-neuronal cell number (fixed effect), and litter (random effect). The mediator model was total cell/neuron/non-neuronal cell number regressed on postnatal treatment (fixed effect), sex (fixed effect), and litter (random effect). Confidence intervals (CI’s) for mediation analyses were bootstrapped (DiCiccio and Efron 1996) 100,000 times to guarantee robust analysis given our sample sizes of either 27 observations (total cell number) or 18 observations (total neuron/non-neuronal cell number).

Results

Blood alcohol concentration

Animals sacrificed on PD7 and PD11 achieved similar peak BACs when measured on PD7 (β = −33.48 ± 54.47, t(23.00) = −0.615, p = 0.545). Animals sacrificed on PD72 achieved significantly lower BACs on PD7 than animals sacrificed on PD7 or PD11 (β = −139.58 ± 54.47, t(23.00) = −2.563, p = 0.017). There was no influence of sex on peak BAC (β = −43.88 ± 54.47, t(23.00) = −0.806, p = 0.429). Mean peak BACs, along with mean weights, are reported in Supplemental Material Table 1.

The subset of animals used for cell type analysis (sacrificed at PD72) had a mean peak BAC of 332.65 (±33.6) mg/dl (mean ± SEM), and did not significantly differ from the BACs of animals sacrificed on PD7 (β = 32.52 ± 45.39, t(19.00) = 0.717, p = 0.0.482) or PD11 (β = −0.96 ± 45.39, t(19.00) = −0.021, p = 0.0.983). There were still no differences between BACs from male and female pups in this subset of animals (β = −57.08 ± 46.98, t(19.00) = −1.215, p = 0.239).

Alcohol exposure on postnatal day 7 causes apoptotic cell death in Re

Weight

There was no weight difference among postnatal treatment groups prior to manipulation (p’s > 0.650). All animals gained weight between the morning of PD7 and time of perfusion on the evening of PD7 (β = 0.80 ± 0.20, t(24.00) = 3.930, p = 0.001). There was an interaction between age and postnatal treatment such that, while sham intubated (SI) animals did not differ in weight at the start of the experiment, SI animals weighed significantly greater than suckle control (SC) and AE at 12 hours after intubation (β = 1.06 ± 0.29, t(24.00) = 3.682, p = 0.001). There was no influence of sex either prior to manipulation, or at time of perfusion on PD7 (p’s > 0.760).

Volume of Re

A representative outline of Re, and demonstrations of apoptotic bodies in SI and AE tissue can be found in Figure 2, alongside mean unbiased stereological estimates of Re volume and estimated apoptotic cell number, respectively. While volume of Re did not differ between SC and SI animals (β = −0.207 ± 0.345, t(2.56) = −0.600, p = 0.597), there was an interaction between AE and sex such that AE females had greater Re volume than all groups (β = 0.910 ± 0.400, t(22.57) = −2.193, p = 0.039), while AE males did not differ from any control groups (β = 0.628 ± 0.361, t(2.96) = 1.741, p = 0.181).

Apoptotic cell death in Re

AE animals displayed a significant increase in apoptotic cell number at 12 hours postexposure (β = 33835 ± 4531, t(4.96) = 7.467, p = 0.001) relative to SI and SC animals. SC and SI animals displayed similar levels of cell death (β = −288 ± 4277, t(4.47) = −0.067, p = 0.949). There was no influence of sex on apoptosis in Re (all main effect and interaction p’s > 0.200).

Alcohol exposure on postnatal day 7 reduces cell number in Re after 96 hours

4 days after AE insult (i.e., PD11), apoptotic bodies were minimally present in the Re of animals from all 3 treatment groups. This suggests that AE-induced bursts of apoptosis are transient and return to control levels by PD11, and that naturally-occuring apoptosis is also reduced between PD7 and PD11.

Weight

There was no weight difference between postnatal treatments (p’s > 0.380), nor sexes (β = 0.24 ± 0.65, t(34.10) = 0.370, p = 0.714), on PD7. While all animals clearly increased in weight from PD7 to PD11 (β = 7.34 ± 0.44, t(23.00) = 16.742, p < 0.001), there was an interaction between AE and age, indicating that AE pups gained less weight than SC or SI pups between PD7 and PD11 (β = −1.98 ± 0.62, t(23.00) = −3.194, p = 0.004). Due to this weight difference at PD11, LMM analysis of cell number and volume on PD11 will include weight on PD11, to statistically adjust for potential weight differences as a covariate.

Total cell number in Re

Analysis of unbiased stereological estimates of total cell number in Re on PD11 (Figure 3) utilized postnatal treatment, sex, and weight on PD11 (as a covariate) as fixed effects, and litter as a random effect. AE animals displayed significant cell loss in Re (β = −143746 ± 49029, t(14.00) = −2.932, p = 0.011), while SI did not display cell loss (β = −17613 ± 47086, t(14.00) = 0.374, p = 0.714). There was no influence of sex (all main effect and interaction p’s > 0.480).

Figure 3: Alcohol exposure (AE) on postnatal day 7 causes cell loss in thalamic nucleus reuniens in postnatal day 11 rat brain.

Figure 3:

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A representative image of cresyl violet-stained tissue (panel 3A; scale bar = 1mm) demonstrates a typical contour of reuniens. Due to the absence of sex differences or sex-based interactions (p’s > 0.480), data in 3B are collapsed across sex. AE caused a significant reduction in cell number at 4 days post-AE (p = 0.011). There was no significant impact of SI (p = 0.714). Graphs present raw data points (in grey) with black bars representing mean ± SEM for that group. Groups with similar letters over them indicate that those groups did not significantly differ.

Alcohol exposure on postnatal day 7 reduces neuron number in reuniens into adulthood

Weight

There were no influences of postnatal treatments on animal weight on either PD7 or PD72 (all main effect and interaction p’s > 0.270). While all animals clearly increased in weight from PD7 to PD72 (β = 238.98 ± 11.92, t(45.94) = 20.051, p < 0.001), there was an interaction between sex and age, indicating that males weighed more than females at PD72 (β = 131.64 ± 16.86, t(45.94) = 7.810, p < 0.001), but not at PD7 (β = 0.19 ± 11.97, t(46.80) = 0.016, p = 0.998).

Total Cell Number

Mean unbiased stereological estimates of total volume of Re and total cell number in Re can be found in Figure 4, alongside a representative contour of Re in a cresyl violet-stained brain section (Figure 4A). While SI animals displayed significant cell loss in Re (β = −40453 ± 15804, t(9.49) = −2.560, p = 0.029), AE was associated with a significantly greater cell deficit relative to SC and SI groups (β = −78821 ± 16743, t(13.10) = −4.708, p < 0.001). There was no sex effect on cell loss (all main effect and interaction p’s > 0.310).

Figure 4: Alcohol exposure (AE) on postnatal day 7 results in permanent neuron loss in thalamic nucleus reuniens, while intubation independently reduces non-neuronal cell number relative to typically developing control animals.

Figure 4:

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A representative image of cresyl violet-stained tissue (panel 4A; scale bar = 1mm) demonstrates a typical contour of reuniens. Due to the absence of sex differences or sex-based interactions (p’s > 0.131), data are collapsed across sex. Significant volume and cell loss was observed in Re of AE animals (panel 4B). Less severe cell loss was also observed in sham intubated (SI) animals (4B, right). Follow-up analysis with NeuN-labeled tissue (panel 4C; counterstained with Pyronin Y; scale bar = 1mm) revealed that alcohol uniquely reduced neuron number in Re (panel 4D, left) while intubation reduced non-neuronal cell number in both the SI and AE groups (panel 4D, right). Graphs present raw data points (in grey) with black bars representing mean ± SEM for that group. Groups with similar letters over them indicate that those groups did not significantly differ.

Cell type-specific alterations to reuniens

Immunocytochemical labeling of neurons using antibodies against the NeuN protein can be observed in figure Figure 4C. Analysis of neuron number in Re (Figure 4D) indicated that neuron number in adult AE animals was reduced relative to both SI and SC adult controls (β = −24242 ± 5456, t(4.83) = −4.443, p = 0.007). SC and SI groups did not differ from each other in total neuron (NeuN+) number (β = −3827 ± 5456, t(4.16) = −0.784, p = 0.475). In contrast, both SI and AE groups displayed a significant reduction in non-neuronal (total cell # - NeuN+) cell number (Figure 4D) relative to the SC group (β = −46361 ± 19743, t(12) = −2.348, p = 0.037). The number of non-neuronal cells did not differ between SI and AE groups (β = −46786 ± 22074, t(12) = −2.119, p = 0.056).

Mediation analysis indicated that AE reduced Re volume (estimate = −0.436, 95% CI = −0.747 to −0.126, p = 0.007), an effect that was mediated indirectly by reductions in total cell (i.e., neuronal and non-neuronal) number (estimate = −0.588, 95% CI = −0.997 to −0.243, p < 0.001). In contrast, there was no direct effect of alcohol on Re volume independent of cell loss (estimate = 0.152, 95% CI = −0.252 to 0.553, p = 0.464). A graph of these data plotted against each other can be observed in Figure 5A, while a visualization of the mediation relationship (with regression coefficients provided in units of # of cells or mm3) can be found in Figure 5B. The mediation for the effect of PD7 alcohol exposure on Re volume mediated by neuron loss alone was not significant (estimate = −0.271, 95% CI = −0.737 to 0.200, p = 0.250), nor was the mediation for non-neuronal cells (estimate = −0.272, 95% CI = −0.708 to 0.170, p = 0.215), suggesting that AE-induced reduction in Re volume is driven by a general loss of cells rather than the absence of any particular subtype.

Figure 5: Alcohol exposure on Postnatal day 7 decreases volume of thalamic nucleus reuniens (Re) through cell loss.

Figure 5:

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Raw data points for volume and total cell number estimates for each animal (5A). Red circles indicate SC animals, green triangles indicate SI animals, and hollow blue boxes indicate AE animals. Mediation analysis revealed that AE was associated with a significant reduction in Re volume (5B; p = 0.007). This reduction in Re volume was mediated by a reduction in cell number (p < 0.001), rather than a direct influence of AE on volume (p = 0.464, value in parentheses in diagram). Values in 5B are raw (non-standardized) coefficient values from mediation analysis, and are measured in either total cell number (for “Re Cell #”) or mm3 (for “Re Volume”), † not significant, ** p < 0.010, *** p < 0.001

Discussion

The current study sought to characterize the short- and long-term impact of single-day binge alcohol exposure on cell number and volume in thalamic nucleus reuniens. We observed robust alcohol-induced cell death in Re on PD7, and persistent alcohol-induced reduction in cell number across the lifespan (i.e., PD11 and PD72), which was caused by a loss of neurons. These data suggest that Re is highly vulnerable to bingelike alcohol exposure regardless of the duration of exposure.

There was no influence of biological sex on apoptosis or cell number in Re, suggesting a similar mechanism by which AE induces persistent neuroanatomical damage in Re of both the male and female brain: through apoptotic cell loss. Our observation that neuronal number was reduced in Re of AE animals relative to both control groups suggests that neurons are particularly vulnerable to insult as this early period, as observed in the current study following a single-day alcohol exposure, as well as in our previous report (Gursky, Savage et al. 2019), following 6-day developmental alcohol exposure. Observing comparable outcomes following either single- or six-day AE suggests that Re is highly vulnerable to alcohol-induced damage. Due to practical limitations, the current study was unable to longitudinally examine the relationship between cell death and future cell number in a single brain. While the current study suggests that AE-induced apoptosis results in neuron loss, co-administration of an anti-apoptotic agent with alcohol exposure would be necessary to establish causality of increased apoptosis on neuron loss.

A surprising reduction in total Re cell number (but not neuronal number) was observed in sham intubated animals on PD72, while no intubation-related increase in apoptosis was found on PD7. This suggests that even a single day of intubation can lead to altered brain development throughout life (Boschen, Ruggiero et al. 2016), an issue often raised in alcohol literature (Kelly and Lawrence 2008). This intubation-related reduction in cell number was exclusively non-neuronal, which suggests that the stress of intubation likely alters the survival or proliferation of glial cells, a process that occurs dissociably from the alcohol-related reduction in neuron number.

While we observed similar magnitude decrease in non-neuronal cell number in the AE and SI groups at PD72, it is possible that such cell deficit was caused by a reduction in different phenotypes types of glial cells between the two postnatal treatment groups, as non-neuronal cells are a very diverse population in the brain. Such would have to be the case in order for the loss of glia to drive the reduction in Re neuron number of AE animals but not SI animals. This hypothesis deserves significant attention in future studies, with the current study providing an important foundation. Although our data suggest that alcohol-induced apoptosis leads to reduced Re neuron number throughout life, we recognize that alcohol-induced alterations to glial populations may also contribute to the observed neuronal deficit (i.e., that both processes may be contributing to the single observed outcome), as studies in non-human primates have indicated that oligodendrocytes are particularly vulnerable to alcohol-induced apoptosis during a similar neurodevelopmental period (Creeley, Dikranian et al. 2013). Creeley, Dikranian et al. (2013) suggest that any change in either neuronal or non-neuronal apoptosis caused by exposure to teratogens at any point from embryogenesis until well into postnatal life can result in drastic long-term consequences for the damaged structure and those related to it.

The only influence of sex in our data was an increase in volume of Re in female AE animals at 12 hours post-AE. It is well recognized that infiltration of microglia, the resident immune cells of the brain, occurs differently across the sexes (Bilbo and Schwarz 2012). Region- and sex-specific differences in inflammatory function during early development (Schwarz, Sholar et al. 2012) could account for the increase in Re volume observed in females. This hypothesis necessitates further study of neuron-glia interactions in Re throughout development. Our data also suggest that there is minimal cytogenesis in Re after PD11, as the number of cells present in Re of typically-developing animals at PD11 and PD72 were similar. While the period of developmental neurogenesis ends in Re around embryonic day 17, our data assess total cell number, suggesting that glial cells are also present at their adult numbers by the second week of postnatal life.

We propose that Re — the most direct and prominent pathway for information transfer from mPFC to HPC (Cassel and Pereira de Vasconcelos 2015) which remains understudied in the context of neurodevelopmental disorders — is more sensitive than previously expected to alcohol-induced damage in early postnatal life. Gursky, Savage et al. (2019) previously identified that neuron loss following multiple days of alcohol exposure was selective to Re (i.e., was not present in the adjacent rhomboid nucleus of ventral midline thalamus). It is likely that susceptibility of thalamic nuclei is driven by a factor other than developmental age of neurons in the region, as neurons in both Re and the neighboring rhomboid nucleus of thalamus are undergoing neurogenesis at the same time (Altman and Bayer 1979), yet differ in susceptibility to developmental alcohol exposure (Gursky, Savage et al. 2019). The next goal of this topic is to further examine the susceptibility of other thalamic nuclei outside of ventral midline thalamus to early postnatal AE in order to find common features of vulnerable versus resilient nuclei.

Recent research has indicated that Re projections to distinct subregions of mPFC underlie dendritic spine plasticity and stability of certain behavioral tasks (Klein, Cholvin et al. 2019). Further examination into how the loss of neurons in Re relates to connectivity with mPFC and HPC is an essential next step to determining the impact of this newly observed phenomenon on behavioral outcomes in fetal alcohol spectrum disorders.

While we observed differences in BAC on PD7 across animals sacrificed for different time points (specifically, a significantly reduced peak BAC of animals that were used for PD72 cell number analysis), the observed data still support that Re damage can occur following BACs lower than those necessary to induce robust increases in apoptosis. The BACs in the subset of animals used for neuron analysis (where AE-induced neuron loss was observed) did not significantly differ from the BACs of animals sacrificed at PD7 or PD11. If anything, this decrease in BAC in adult cell number analysis supports our belief that Re is highly vulnerable to alcohol-induced cell loss in rodent models of third trimester alcohol exposure.

In conclusion, our findings expand the body of literature consisting of only one other study examining the effects of third trimester-equivalent alcohol exposure on ventral midline thalamus (Gursky, Savage et al. 2019). It adds to the growing body of literature informing about the potential danger of a single-day alcohol exposure for an unborn child. The prevalence of executive functioning deficits in humans diagnosed with FASD make this functional neuroanatomical circuit long overdue for examination.

Supplementary Material

1

Highlights.

  • Early postnatal alcohol exposure causes apoptotic cell death in nucleus reuniens (Re)

  • Single-day postnatal AE causes permanent neuron loss in Re

  • Alcohol-induced changes in Re volume are mediated by alcohol-induced cell loss

  • Intubation, a common model of FASD, reduces non-neuronal cell number in Re

Acknowledgements

This research was supported by NIAAA 1 R21 AA026613-01 to AYK, NIH/NIGMS COBRE: The Delaware Center for Neuroscience Research Grant 1P20GM103653-01A1 to AYK, NIAAA R01 AA027269 to AYK, University of Delaware Office of Graduate and Professional Education’s University Doctoral Fellowship to ZHG, and University of Delaware Summer Scholars award to ECS.

Thanks to the undergraduate research assistants that helped with animal generation and tissue preparation. Additional thanks to Dr. Adam Davey for his training on — and assistance with — the statistical analyses performed.

Abbreviations

AE

Alcohol exposure/ Alcohol exposed treatment group (experimental manipulation)

BAC

Blood alcohol content

EF

Executive function

FASD

Fetal Alcohol Spectrum Disorders

HPC

Hippocampus

mPFC

Medial prefrontal cortex

PD

Postnatal day

Re

Thalamic nucleus reuniens

SC

Suckle control (typically-developing control group)

SI

Sham intubation (procedural control group)

Footnotes

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