Assessment of GABA_A α1, α4, and α5 Receptor Subunit Protein Expression in Dorsal and Ventral Hippocampus of Adult Male and Female Long-Evans Rats after Moderate Prenatal Alcohol Exposure

Abstract

Fetal Alcohol Spectrum Disorders (FASD) describe a range of neurobiological and behavioral changes following prenatal alcohol exposure. Previous work has shown that moderate prenatal alcohol exposure (mPAE) can produce alterations to GABAergic neurotransmission in the hippocampal formation (HPF) as well as morphological alterations in GABAergic interneuron expression in adult rats. In the present study, we tested the hypothesis that mPAE differentially alters GABA_A subunit protein expression in dorsal and ventral HPF of naïve adult male and female Long-Evans control and mPAE rats. We focused our attention on the α1, α4, and α5 subunits as they have relatively high densities within the hippocampus and have been linked to hippocampal-dependent learning and memory. Membrane fractions were collected from dorsal and ventral HPF, and western blots were performed to detect levels of GABA_A α1, α4, and α5 subunit protein expression. Overall, we did not detect prenatal treatment group differences suggesting that mPAE does not result in differential GABA_A subunit protein expression in dorsal or ventral HPF of male and female rats. However, the results revealed significantly greater protein expression for α4 and α5 subunits in the dorsal HPF. GABA_A α1 protein expression did not vary significantly across the dorsal and ventral HPF. Our findings support the general conclusion that GABA_A subunit protein expression in the HPF reflects intrinsic regional specialization of GABAergic signaling rather than effects of mPAE.

Introduction

FASD is a multifaceted neurodevelopmental disorder characterized by alcohol exposure during gestation causing aberrant fetal brain development (Felicicchia et al., 2025; Popova et al., 2023) and cognitive impairments involving executive functions, memory, and affective behaviors (Marquardt & Brigman, 2016). The clinical presentation of FASD overlaps with other neurodevelopmental disorders which makes accurate diagnosis challenging. The global prevalence, which is between 1%–5% of children (Waite & Burd, 2023), is likely underestimated in moderate cases of exposure (e.g., mPAE) due to clinical under-recognition.

Previous research has shown that mPAE (~30–100 mg/dL) has been associated with impairments in HPF-dependent memory, alterations to synaptic plasticity neurogenesis, and memory related neural activity (place cells) in the HPF (Choi et al., 2005 Clark et al., 2025; Harvey et al., 2020; Marquardt & Brigman, 2016; Savage et al., 2002; 2010; Takahashi et al., 2021). Developmental ethanol exposure can also cause widespread disruptions in GABAergic circuit development and function in HPF and related regions (Barber et al., 2022; Bird et al., 2018; 2021; Kenton et al., 2020; Lee et al., 2022; Licheri et al., 2023; Madden et al., 2020). Alcohol exposure has been linked to abnormal GABAergic interneuron migration and reductions in parvalbumin-positive interneurons (Delatour et al., 2020; Lee et al., 2022; Marguet et al, 2020; Licheri et al. 2023) and enhanced or diminished GABAergic signaling (Bird et al., 2021; Kenton et al., 2020; Cuzon Carlson et al., 2020; Delatour et al., 2020). The effects of mPAE show sexually dimorphic effects with females demonstrating more variable changes such as enhanced GABAergic signaling (Winchester & Diaz, 2025; Tousley et al., 2025).

It is currently unclear whether mPAE produces modifications to GABA receptor structure. GABA receptors are classified into GABA_A and GABA_B subtypes (Sigel & Steinmann, 2012). GABA_A receptors are ligand gated ion channels that underlie fast inhibitory neurotransmission in the HPF and likely have a role in generating rhythmic oscillations associated with memory (Farrant & Nusser, 2005). GABA_A receptors, which are the primary targets for alcohol in the nervous system (Olsen & Liang, 2017), are assembled from a pool of 19 different subunits, with a majority comprised of α, β, and γ subtypes (Farrant & Nusser, 2005; Sigel & Steinmann, 2012). HPF GABA_A receptors contain a high density of the α subunit, particularly the α5 subunit, and are speculated to play a role in memory functions (Collinson et al., 2002; Serwanski et al., 2006). For instance, reductions in α5 expression have been associated with improved spatial memory in mice (Collinson et al., 2002). Alcohol-induced spatial memory dysfunction has been associated with increases in α5 mRNA expression (Zeng et al., 2019). Similar observations have been made for α1 and α4 GABA_A subunits, as disruptions to these subtypes alter synaptic plasticity and memory (Berry et al., 2008; Shen et al., 2010; Wiltgen et al., 2009). Previous work has also shown that α4 and α5 subunits are found in extrasynaptic receptor assemblies contributing to tonic inhibition and maintaining sparse activity, which are essential to memory related computations by the HPF (Nagarajan et al., 2026).

There is some evidence that alcohol exposure during early development can affect GABA_A subunit expression. For instance, chronic alcohol exposure in young adult rats over a period of 40 days, but not 14 days, increases α4 subunit expression in the HPF (Matthews et al., 1998). Liang et al (2014) reported functional disruptions to α1 subunit containing GABA_A receptors in the HPF of adolescent rats (PND25–35) after prenatal gavage exposure to alcohol. Lastly, Allan et al (1998) reported that neuromodulatory effects on GABA_A receptor-mediated inhibitory neurotransmission were enhanced in the HPF after mPAE, which may be attributable to differences in GABA_A α subunit expression, and may ultimately contribute to synaptic plasticity deficits observed after mPAE.

Here, we tested the hypothesis that HPF GABA_A receptor subunit protein would be elevated after mPAE. We performed western blots to investigate GABA_A α1, α4, and α5 subunit protein expression within the HPF of a rat model of mPAE in which previous studies have characterized impairments in spatial memory, hippocampal synaptic plasticity, and neural representations associated with learning and memory in adult rats (Davies et al., 2023a; Harvey et al., 2020; Sanchez et al., 2025). Although previous work in adult rats has reported that mPAE can produce reductions in parvalbumin-positive GABA interneuron expression in the dorsal HPF (Madden et al., 2020), behavioral studies report deficits in affective behaviors dependent on the integrity of the vHPF (Acosta et al., 2024). We therefore designed our study to investigate whether expression of GABA_A α1, α4, and α5 subunit protein expression in the dHPF and vHPF would be impaired in adult rats after mPAE.

Methods

Breeding Colony and Ethanol Consumption Procedures

The breeding and ethanol drinking paradigm used in the present study were identical to recent studies from our group (Fig. 1) (Acosta et al., 2024; Davies et al., 2023b; Osterlund Oltmanns et al., 2022; Schaeffer et al., 2025). Briefly, adult female Long-Evans rat breeders (Envigo Corporation, Indianapolis, IN) were single-housed at 22°C and maintained on a reverse 12h dark/12h light schedule and were provided PMI Picolab 5L0D laboratory rodent diet (LabDiet Incorporated, St Louis, MS) and tap water ad libitum. Female Long-Evans rat breeders were acclimated to ethanol by providing a solution of 0.067% saccharin in tap water with 0% ethanol on day 1 and 2, saccharin water with 2.5% ethanol on days 3 and 4, and saccharin water with 5% ethanol on day 5 and thereafter. The saccharin and ethanol mixtures were provided for 4h each day from 10:00–14:00 h for at least two weeks. Daily ethanol consumption was measured for each female rat breeder. Following two weeks, females who drank at levels less than one standard deviation below the entire group mean were removed from the study. The remaining breeders were assigned to either a saccharin (Control) or 5% ethanol (Ethanol) drinking group. Females were weighed to obtain a pre-pregnancy weight, then housed with male breeders until a vaginal plug was present to confirm pregnancy. Ethanol consumption did not occur during breeding. Once pregnant, dams were given access to saccharin water containing either 0% (v/v) or 5% (v/v) ethanol for four hours each day (10:00–14:00). The volume of saccharin water provided to the Control group was matched to the mean volume of ethanol saccharin water consumed by the Ethanol group. The volume of ethanol consumed was recorded for each rat dam as grams of ethanol per kilogram of body weight per day. At birth, daily ethanol consumption was discontinued, and the litters were weighed and culled to 10 pups. Offspring were weaned at postnatal (PD)21–24 and pair-housed (see section below). To minimize potential litter effects in the present study, only one or two female or male rats were used from each litter.

Figure 1.

Experimental timeline for mPAE and tissue collection. Adult female rats were acclimated to a voluntary ethanol drinking paradigm (increasing concentrations of ethanol) during the pre-pregnancy phase. Following mating, pregnant dams were assigned to either saccharin (Control) or 5% ethanol (EtOH) groups and maintained on their assigned solution for throughout gestation (GD1–GD21). HPF tissue was collected at postnatal day 217–229 for fluorescent western blot analysis.

Subjects

A total of 24 Long-Evans rats (Control: n=12; 6 male and 6 female; Ethanol: n=12; 6 male and 6 female) were obtained from the University of New Mexico Health Sciences Center (UNMHSC) Animal Resource Facility (ARF) (see breeding protocol above). Following weaning (PD21–24), all rats were pair-housed in standard plastic cages on a reverse 12h dark/12h light schedule at a room temperature of 22 °C with the PMI Picolab 5L0D laboratory rodent diet (LabDiet Incorporated, St Louis, MS) and water provided ad libitum. The Institutional Animal Care and Use Committee (IACUC) of the UNMHSC approved all experimental procedures for the current study.

Membrane preparation and GABA_A western blotting

The experimenters conducting the membrane preparations and western blot analyses were blinded to the experimental condition of the tissues processed. Adult male and female offspring (PND217–229) were anesthetized with 250 mg/kg ketamine intraperitoneal injections, sacrificed, and the hippocampal formation (HPF) removed and was separated into two approximately equal dorsal (dHPF) and ventral (vHPF) halves. The tissue was weighed and homogenized in a glass Dounce homogenizer in homogenization buffer (320 mM sucrose, 10 mM Tris HCl, 1 mM Na₃VO₄, 5 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 × phosphatase inhibitor cocktail 3 (Sigma-Aldrich, Inc. St Louis, MO) and 1 × SigmaFast protease inhibitor (Sigma-Aldrich, Inc.) with six up and down strokes each of a loose-fitting and then a tight-fitting pestle. The homogenate was centrifuged at 1000g for 6 min at 4°C and then the S1 supernatant was collected into a second tube and centrifuged at 10,000g for 15 min at 4°C to collect the P2 pellet. The P2 pellet was resuspended in homogenization buffer and aliquoted. Samples were then flash-frozen in liquid nitrogen and stored at −80°C until assay. Proteins were quantified using a BCA Protein Assay Kit (Pierce, Rockford, IN) according to manufacturer's instructions. The following primary antibodies were used: rabbit anti-GABA A α1 (ab33299, Abcam Inc. Waltham, MA; diluted 1:1000), rabbit anti-GABA A α4 (NB300–193, Novus Biologicals, LLC, Centennial, CO; diluted 1:1000), rabbit anti-GABA A α5 (224–503, SYSY antibodies, Germany; diluted 1:1000) and the reference protein, mouse anti-Na⁺/K⁺ ATPase (A5-s, DSHB, Iowa City, IA; diluted 1:10,000 or 1:20,000).

Based on preliminary tissue concentration studies to confirm linear optical density measures for each protein of interest in untreated control rats (data not shown), 30 or 40 μg of total protein were loaded per lane on a NuPAGE^™ 4–12% Bis-Tris 12-well mini protein gel (Thermo Fisher Scientific, Waltham, MA) in triplicate lanes and were run at 150–155 V for 2.5 h. Protein samples were denatured at 70°C and samples run with MOPS SDS buffer (Thermo Fisher Scientific). The proteins were transferred onto methanol-activated PVDF membranes using NuPAGE^™ transfer buffer (Thermo Fisher Scientific) with methanol and NuPAGE^™ antioxidant (Thermo Fisher Scientific) and run at 100 V for 1 h. The membranes were blocked using LI-COR Intercept^™ blocking buffer (PBS) (LI-COR, Lincoln, NE) at room temperature for 1 h, then incubated with the primary antibodies at 1:1000 dilution, overnight at 4°C in the LICOR Intercept blocking buffer (PBS) plus 0.1% Tween-20. Membranes were then washed three times for 10 min in PBS-Tween 20 (PBS-T), incubated in secondary antibodies (LI-COR 680RD goat anti-mouse, 1:40,000; and 800CW donkey anti-rabbit, 1:15,000;) in LI-COR Intercept blocking buffer (PBS) plus 0.1% Tween-20 and 0.01% SDS for 1 h at room temperature. Membranes were washed three times for 10 min in PBS-T and an additional 5 min in PBS before scanning. Membranes were scanned and analyzed using the LI-COR Odyssey Infrared Imaging System and quantified in Image Studio^™ 5.1. Briefly, after setting a blot’s background intensity in an area with no fluorescence, boxes were drawn around each fluorescent band and the background intensity subtracted from each band of interest to provide an integrated signal intensity.

With the blots, we used a ratio of GABA subunit/ATPase to control for inter-blot variability. Once all blots are normalized to a housekeeping protein (here ATPase), you can directly compare the values across different membranes. We also looked at the mean integrated intensity for ATPase which was similar across groups (Table 2). Subsequently, the optical densities of each GABA_A subunit were normalized to the reference protein, Na⁺/K⁺ ATPase.

Statistical Analysis

A mixed-design analysis of variance (ANOVA) was conducted for each GABA_A subunit (α1, α4, and α5), comparing dHPF and vHPF (within-subjects), with treatment (Control and Ethanol) and sex as between-subjects factors. Partial eta squared (η²p) values are reported as a measure of effect size. Analyses were conducted using jamovi (The jamovi project, 2025).

Results

Paradigm Outcome Measures

Supplemental Table 1 presents a summary of the mPAE paradigm outcome measures for the rat dams in control and mPAE groups. The mean ethanol consumption by the mPAE group was 2.32 g/kg which, when coupled with the 5L0D diet, produces a maternal blood ethanol concentration (BEC) of 46.0 + 3.2 mg/dL (Davies et al., 2023b). There were no significant effects on maternal weight gain, litter size or offspring birthweight (Supp Table 1).

ATPase Expression in the Hippocampus

Figure 2 shows representative western blots from dHPF or vHPF for GABA_A α1, α4, and α5 subunits. The apparent kD values for each of the four proteins analyzed was consistent with published molecular weights for each protein. Each blot contained triplicate samples of either dHPF or vHPF from three of the four experimental conditions. The three conditions selected for each blot was balanced across the total of eight blots required to analyze all 24 samples for a given protein in each region.

Figure 2.

Representative fluorescent western blot images of GABA_A α1 (A), α4 (B), and α5 (C) subunits in dHPF) and vHPF) homogenates (green band). ATPase was used as the reference protein (red band). The x-axis corresponds to the treatment group and sex of the animals. CF = Control Female; CM = Control Male; EF = Ethanol Female; EM = Ethanol Male.

Supplemental Table 2 summarizes the mean and standard error for ATPase measures (averaged across α1, α4, and α5 subunits) for treatment, sex, and HPF region. Inspection of the means indicate that ATPase measures were generally similar across dHPF and vHPF regions but were slightly higher in vHPF. A mixed-design ANOVA comparing ATPase across dHPF and vHPF regions did not indicate group differences (ps > 0.51), but detected a significant dHPF versus vHPF difference (F(1, 20) = 5.94, p = .024, η²_p = .229). The ANOVA did not detect significant interactions (ps > 0.40).

GABA_A Receptor Subunit Expression

Figure 3 shows ratios of GABA_A subunit expression relative to the reference protein, ATPase, across the dHPF and vHPF for Control and Ethanol rats. A mixed-design ANOVA determined that α1 subunit expression (Fig. 3A) did not significantly differ by region (F(1, 20) = 0.63, p = .437, η²_p = .030), treatment (F(1, 20) = 0.28, p = .606, η²_p = .014), or sex (F(1, 20) = 1.05, p = .317, η²_p = .050). None of the two-way interactions were significant: sex × treatment (F(1, 20) = 0.07, p = .788, η²_p = .004), sex × region (F(1, 20) = 0.19, p = .666, η²_p = .010), treatment × region (F(1, 20) = 0.31, p = .581, η²_p = .015), or the three-way interaction (F(1, 20) = 0.69, p = .416, η²_p = .033). In contrast, α4 subunit expression (Fig. 3B) exhibited a significant main effect of region (F(1, 20) = 62.20, p < .001, η²_p = .757), with higher expression in dHPF compared to vHPF. However, neither treatment (F(1, 20) = 0.01, p = .923, η²_p < .000) nor sex (F(1, 20) = 0.42, p = .523, η²_p = .021) had a significant effect, and all interactions were non-significant: sex × treatment (F(1, 20) = 0.06, p = .815, η²_p = .003), sex × region (F(1, 20) = 0.59, p = .450, η²_p = .029), treatment × region (F(1, 20) = 0.09, p = .770, η²_p = .004), and the three-way interaction (F(1, 20) = 0.15, p = .706, η²_p = .007).

Figure 3.

The effect of mPAE on the ratios of GABA_A α1 (A), α4 (B), and α5 (C) subunit expression in dHPF versus vHPF. Bars represent the mean ± SEM of normalized fluorescent western blot signal intensity, with values expressed relative to the reference protein, ATPase. Control (cyan) and Ethanol (magenta) groups are shown separately for female and male offspring. Each white circle represents one animal (n = 6 per group). (***p < 0.001).

Regional differences in GABA_A subunit expression were independent of prenatal alcohol exposure and sex. The findings support intrinsic regional specialization of hippocampal GABAergic signaling.

A similar pattern was observed in α5 subunit expression (Fig. 3C), which showed a strong regional effect (F(1, 20) = 104.36, p < .001, η²_p = .839), with significantly higher levels in dHPF compared to vHPF. No main effects were found for sex (F(1, 20) = 0.003, p = .959, η²_p =.000) or treatment (F(1, 20) = 0.45, p = .511, η²_p = .022), and all interaction terms were non-significant: sex × treatment (F(1, 20) = 0.56, p = .465, η²_p = .027), sex × region (F(1, 20) = 0.44, p = .514, η²_p = .022), treatment × region (F(1, 20) = 0.02, p = .903, η²_p = .001), and sex × treatment × region (F(1, 20) = 0.43, p = .520, η²_p = .021).

Discussion

The results of the present study point to two general conclusions. First, Control and mPAE rats expressed similar levels of GABA_A α1, α4, and α5 protein within dHPF and vHPF. It is important to note that while GABA_A α1, α4, and α5 subunit expression remained intact in dHPF and vHPF after mPAE, other work has reported that mPAE in male rats can cause high levels of stress hormones in utero, leading to overexpression of GABA_A α1 subunit mRNA in the vHPF (Ardi et al., 2016; 2019) and subsequent upregulation of the β₁ and β₂ GABA_A subunits in the hypothalamus (Lu et al., 2020). These findings may appear paradoxical; however, they can be reconciled by considering a model of presynaptic dysfunction. Cuzon Carlson et al. (2020) investigated the effects of mPAE on goal-oriented behavior and memory and found that upregulation of endocannabinoid expression chronically weakened parvalbumin interneuron synaptic projections with medium spiny neurons in the dorsolateral striatum, suggesting a presynaptic mechanism underlying cognitive dysfunction. Similarly, a study by Delatour et al. (2020) found increased frequency of spontaneous and miniature IPSCs and EPSCs, with an enduring amplitude across control and ethanol-exposed groups. These findings point to alterations in presynaptic release probability or synapse number, rather than postsynaptic receptor function.

The present study employed adult offspring in an age range similar to our prior studies reporting alterations in parvalbumin-positive GABAergic interneurons (Madden et al., 2020) and spatial memory impairments (Sanchez et al., 2025; Savage et al., 2002). While we did not observe changes in expression in adult offspring after mPAE, it is possible that changes may have occurred earlier in development followed by some compensatory alterations that normalized receptor subunit expression with maturation. Alternatively, changes in GABA_A subunit expression in dHPF or vHPF may require higher levels of alcohol exposure (current study: ~46 mg/dL; Supp Table 1) or extended exposure during development. Matthews et al (1998) exposed young adult rats to 40 days of a chronic liquid ethanol diet and reported increases in α4 subunit expression in the HPF. However, changes in α4 expression were not observed if rats were exposed for 14 days. Interestingly, α1, α2, α3, β2/3, or γ2 subunit expression in the HPF were unaffected by 40- or 14-day exposure durations. Thus, this previous work suggests that the duration and/or the dose of alcohol used in the present study is likely insufficient to induce GABA_A α1, α4, and α5 subunit expression changes, but also unlikely to induce changes to other GABA_A receptor subunits. Further, an additional factor that may have impacted GABA receptor protein expression was the maternal daily four-hour consumption of 0.067% saccharin water during gestation. While both prenatal treatment groups consumed equal quantities of saccharin, a recent study (Pacheco-Sánchez et. al., 2026) reports that prenatal saccharin exposure altered the mRNA expression of some glutamate and GABA_A receptor subunits in frontal cortex, though GABA α1 expression was not affected.

It is also important to consider that findings of mPAE effects can vary considerably across HPF subregion (CA subfield and/or dentate gyrus). Madden et al (2020) reported a sex-specific reduction in CA3 and dentate gyrus parvalbumin neurons after mPAE. In addition, α4 subunit expression is prominent in the dentate gyrus while α5 is found in CA pyramidal cell layers (Farrant & Nusser, 2005). Thus, it is possible that analyses sampling from all hippocampal subfields may have masked alterations is specific subregions. Related limbic regions may also be affected. For instance, studies have reported excessive inhibition in the dorsolateral striatum (Delatour et al., 2020; Cuzon Carlson et al., 2020), while other studies have shown hyperexcitation in areas such as the paraventricular nucleus after PAE (Lu et al., 2020). Further research would benefit from exploring alterations to GABA_A subunits within HPF subregions and in related limbic circuits.

This absence of sex-specific PAE differences in GABA_A α1, α4, and α5 protein expression within dHPF and vHPF contrasts with a substantial body of research demonstrating sexually dimorphic outcomes of PAE at both the behavioral and physiological level. Mouse and rat-model PAE studies have reported sex-specific alterations in neuronal excitability, striatal GABAergic signaling, and interneuron composition (Cuzon Carlson et al., 2020; Tousley et al., 2025, Licheri et al., 2023; Madden et al., 2020; Kenton et al., 2020; Takahashi et al., 2021). The absence of sex-specific differences in our study suggests that sex-based effects may be context-dependent, influenced by factors such as exposure paradigms, alcohol dosage which is relatively lower in the present study, or the developmental stage at which animals were exposed and/or tested. For instance, a study by Winchester and Diaz (2025) exploring neurobiological changes to the amygdala following mPAE, exposed mPAE neurons to ethanol in vitro and electrophysiological responses were recorded. Winchester and Diaz found that mPAE male central amygdala neurons showed blunted responses to acute ethanol application, whereas mPAE female neurons were largely unresponsive at lower concentrations (44mM and 66mM). However, female central amygdala neurons exhibited significantly enhanced GABA_A-mediated sIPSCs at the highest concentration tested (88mM), indicating a sex- and dose-dependent sensitivity to ethanol following prenatal exposure. It is important to note that Winchester and Diaz (2025) peak BECs of 108.3 mg/dL while the procedures in the present study produce lower BECs at ~46 mg/dL (Davies et al., 2023b). These findings suggest that sex-specific effects are more pronounced at higher concentrations of ethanol and absent or present more subtly at lower doses.

A second conclusion is that α4 and α5 protein expression was significantly higher in the dHPF relative to the vHPF. In contrast, expression of α1 protein was similar across dHPF and vHPF in each group. There was also an appreciable difference in ATPase measures across the two HPF regions with slightly higher measures in vHPF compared to dHPF (Supp Table 2). The findings are consistent with previous observations of distinct anatomical, physiology, and gene markers between dHPF and vHPF (Fanselow & Dong, 2010). While dHPF has been linked to spatial learning and memory, vHPF has been associated with affective behaviors (Fanselow & Dong, 2010). Further, studies of the firing characteristics of place cells have shown that neurons in the dHPF exhibit greater spatially selective firing compared to vHPF (Kjelstrup et al., 2008; Jung et al., 1994). Few studies have investigated GABA_A α1, α4, and α5 protein expression across dHPF and vHPF, however, it is noteworthy that α4 and α5 subunits are almost exclusively found in extrasynaptic receptor assemblies which contribute to tonic inhibition and are critical to preserving sparse activity in the HPF (Nagarajan et al., 2026). The greater α4 and α5 protein expression in dHPF may therefore contribute to the selective spatial firing by place cells in dHPF. Consistent with the present study, a previous report using male Wistar rats (PND35–45) showed a similar decrease in CA1 and dentate gyrus α4 mRNA in vHPF (Sotiriou et al., 2005). However, this same study reported that CA1, CA3, and dentate gyrus α1 mRNA was reduced in vHPF while CA1 α5 mRNA was increased in vHPF. The study also found that α1 protein expression was reduced in vHPF. It is important to note that other work has reported increases in expression of vHPF α1 mRNA only after stress, but with similar mRNA expression across dHPF and vHPF in unstressed animals (Ardi et al., 2019). Thus, more work is needed to understand the underlying mechanism of the current and previous findings.

In summary, we report that GABA_A α1, α4, and α5 subunit protein did not vary between Control and mPAE rats and did not vary with sex. However, we identified higher levels of GABA_A α4 and α5 subunit protein in the dHPF when compared to vHPF. These results suggest that mPAE does not alter the distribution of GABA_A α1, α4, and α5 subunit protein expression in the HPF, but reflects intrinsic dorsal-ventral specialization.