
==== Front
Neurobiol Stress
Neurobiol Stress
Neurobiology of Stress
2352-2895
Elsevier

S2352-2895(24)00061-4
10.1016/j.ynstr.2024.100665
100665
Articles from the Special Issue on Stress and Alcohol; Edited by Barbara Mason and Marisa Roberto
Elevated GABAergic neurotransmission prevents chronic intermittent ethanol induced hyperexcitability of intrinsic and extrinsic inputs to the ventral subiculum of female rats
Bach Eva C. ebach@wakehealth.edu
⁎
Weiner Jeff L.
Department of Physiology and Pharmacology, Wake Forest University, School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA
⁎ Corresponding author. PTCRC, 115 S. Chestnut Street, Winston-Salem, NC, 27101, USA. ebach@wakehealth.edu
10 8 2024
9 2024
10 8 2024
32 1006655 12 2023
31 7 2024
4 8 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
With the recent rise in the rate of alcohol use disorder (AUD) in women, the historical gap between men and women living with this condition is narrowing. While there are many commonalities in how men and women are impacted by AUD, an accumulating body of evidence is revealing sex-dependent adaptations that may require distinct therapeutic approaches. Preclinical rodent studies are beginning to shed light on sex differences in the effects of chronic alcohol exposure on synaptic activity in a number of brain regions. Prior studies from our laboratory revealed that, while withdrawal from chronic intermittent ethanol (CIE), a commonly used model of AUD, increased excitability in the ventral hippocampus (vHC) of male rats, this same treatment had the opposite effect in females. A follow-up study not only expanded on the synaptic mechanisms of these findings in male rats, but also established a CIE-dependent increase in the excitatory-inhibitory (E-I) balance of a glutamatergic projection from the basolateral amygdala to vHC (BLA-vHC). This pathway modulates anxiety-like behavior and could help explain the comorbid occurrence of anxiety disorders in individuals suffering from AUD. The present study sought to conduct a similar analysis of CIE effects on both synaptic mechanisms in the vHC and adaptations in the BLA-vHC pathway of female rats. Our findings indicate that CIE increases the strength of inhibitory neurotransmission in the vHC and that this sex-specific adaptation blocks, or at least delays, the increases in intrinsic vHC excitability and BLA-vHC synaptic transmission observed in males. Our findings establish the BLA-vHC pathway and the vHC as important circuitry to consider for future studies directed at identifying sex-dependent therapeutic approaches to AUD.

Keywords

Anxiety disorder
Alcohol use disorder
Chronic intermittent ethanol
GABAergic
Hyperexcitability
Electrophysiology
Abbreviations

CIE Chronic intermittent ethanol exposure

AUD Alcohol use disorder

BLA Basolateral Amygdala

vSub Ventral subiculum

vHC Ventral hippocampus

E-I Excitatory to inhibitory

ChR2 Channelrhodopsin

BEC Blood ethanol concentrations

oEPSC Optically evoked excitatory postsynaptic currents

oIPSC Optically evoked inhibitory postsynaptic currents

moEPSC Monosynaptic optically evoked excitatory postsynaptic currents

moIPSC Monosynaptic optically evoked inhibitory postsynaptic currents

sEPSC spontaneous excitatory postsynaptic currents

sIPSC spontaneous inhibitory postsynaptic currents

PPr Paired-pulse ratios

ISI Interstimulus interval

TTX Tetrodotoxin

PTX Picrotoxin

Handling Editor: Rita Valentino
==== Body
pmc1 Introduction

Although alcohol use disorder (AUD) has historically been thought of as a disease that predominantly impacts men, sex differences in the rates of binge drinking and the prevalence of AUD have been steadily declining. This phenomenon has been driven by an increase in these problematic behaviors in women, given that rates of both binge drinking and AUD have remained relatively steady in men (Giacometti and Barker, 2020; Grant et al., 2017; Guinle and Sinha, 2020; Han et al., 2017; White et al., 2015). Considering that women who exhibit risky drinking behaviors often develop more severe alcohol-related pathologies than men (Erol and Karpyak, 2015), this trend is particularly concerning. The reasons for the sex-dependent divergence in AUD trends have remained largely conjecture due, at least in part, to a historical dearth of clinical and preclinical studies conducted in women or female experimental subjects. As this serious omission is now finally being corrected, both basic and clinical studies are discovering robust sex differences in neurobiological and associated behavioral adaptations associated with alcohol exposure that may argue for a need to consider sex-specific therapeutic approaches to combat AUD (Flores-Bonilla and Richardson, 2020).

Despite the differences noted above, both men and women suffering from AUD are often diagnosed with comorbid anxiety-, depressive and post-traumatic stress disorders. These conditions can both precipitate and exacerbate AUD (Gilpin and Weiner, 2017; Guinle and Sinha, 2020). Moreover, dysregulation of stress circuitry plays an integral role in prominent theories of AUD (Koob and Volkow, 2016). Given the close link between AUD, stress, and these other mental health disorders it has come as no surprise that many of the brain regions and associated pathological adaptations driving these conditions are shared. The basolateral amygdala (BLA) and the ventral hippocampus (vHC) are two such interconnected brain regions (Almonte et al., 2017; Blaine and Sinha, 2017; Chappell et al., 2013; Grace et al., 2021; Rau et al., 2015; Silberman et al., 2009).

Studies in male rats have consistently shown evidence for hyperexcitability of BLA pyramidal neurons in response to ethanol dependence and a variety of chronic stress models (Blume et al., 2019; Christian et al., 2012; Lack et al., 2007; Rau et al., 2015). The picture of alcohol dependence- and/or anxiety-driven BLA adaptations in female rats has proven more complex. Female rats undergoing withdrawal from chronic intermittent ethanol vapor exposure (CIE), a commonly used model of AUD, initially suggested that females develop hyperexcitability in principal neurons of the BLA similar to their male counterparts but that they simply require more prolonged exposure to CIE (Morales et al., 2018). Follow-up studies, however, have indicated that only a subset of BLA projections neurons experience hyperexcitability driven by CIE while others remained unaffected (Price and McCool, 2022). In line with sex-dependent BLA adaptations, evidence in the clinical population has revealed a reduction in BLA volumes in abstinent alcohol dependent men but not women (Grace et al., 2021). Moreover, at least one group has reported that a rodent model of chronic stress actually leads to a reduction in BLA pyramidal cell neuron excitability in females, possibly as a consequence of sex- and estrous cycle-dependent differences in baseline activity measures (Blume et al., 2017, 2019).

The BLA is thought to regulate anxiety via monosynaptic projections to a variety of cortical and subcortical structures (Felix-Ortiz et al., 2013; Felix-Ortiz et al., 2016; Kim et al., 2013; Pi et al., 2020; Vantrease et al., 2022). Recent work has shown that an excitatory projection from the BLA to the vHC can modulate anxiety-like behaviors and consumatory behaviors of ethanol (Bach et al., 2023; Felix-Ortiz et al., 2013; Pi et al., 2020). Moreover, models of acute and chronic stress promote intrinsic vHC hyperexcitability, although all of these studies have been limited to male subjects (Almonte et al., 2017; Chang and Gean, 2019; Chang et al., 2015). The frequent comorbidity between AUD and anxiety disorders prompted our prior studies looking at synaptic plasticity induced by CIE in the vHC and BLA-vHC of male and female rats. These studies revealed that both sexes exhibited anxiety-like behaviors during acute withdrawal from CIE, although these phenotypes were less pronounced in females. Moreover, this same treatment led to increased extracellular neural excitability in the CA1 of the vHC in males while reducing this measure in females (Bach et al., 2021; Ewin et al., 2019). In a follow up study, looking at individual neurons in the subiculum of the vHC, we confirmed an increase in intrinsic excitatory neurotransmission of this region in male rats. We also showed that CIE increased synaptic excitability in the BLA-vSub pathway (Bach et al., 2021).

In the present study we extended our investigation to explore CIE-dependent neuronal adaptations in vSub excitability and the BLA-vSub pathway in female rats. Using optogenetic approaches we found CIE to have no effect on monosynaptically evoked BLA inputs to the vSub, although polysynaptic driven inputs, engaging intrinsic activity of the vSub, did become strengthened. Importantly, while the excitatory-inhibitory (E-I) balance of the overall BLA-driven input was disrupted (excitation increased) in male rats, it remained unaltered in females. The intrinsic vSub adaptations we previously observed in male rats are likely an important player in these sex-dependent adaptations. In males, CIE increased intrinsic excitatory neurotransmission while inhibition remained unaffected. In female rats we found opposite intrinsic vSub adaptations, with CIE leading to an increase in inhibitory drive and no effect on excitation. Importantly, we also found that blocking this elevated inhibitory drive unmasked BLA-vSub and intrinsic vSub hyperexcitability in female rats (Bach et al., 2021). Taken together our findings support sex-dependent adaptations in response to CIE, particularly in the GABA system, that at least initially decrease susceptibility to hyperexcitation in BLA-vSub pathway-driven and intrinsic vSub inputs of female rats.

2 Methods

2.1 Animals

Female Long Evans rats were purchased from Inotiv, IN and arrived between 9 and 10 weeks. Upon arrival rats were singly housed in clear cages (25.4 cm × 45.7 cm) and maintained on a 12:12 h light dark cycle. Rats had ad libitum access to food (Prolab RMH 3000, LabDiet: PMI Nutrition International, St. Louis, MO) and water throughout the study. Animal care procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Wake Forest University Animal Care and Use Committee. All animal care and use procedures were carried out in compliance with ARRIVE guidelines. A total of 15 CIE and 16 AIR rats were used to complete all electrophysiological studies.

2.2 Stereotactic surgeries

Naïve subjects weighing between 200 and 230 g were anesthetized using sodium pentobarbital (40–50 mg/kg, i. p.). For analgesic purposes animals were injected subcutaneously with 2 mg/kg meloxicam. The scalp was shaved and surgically scrubbed. Rats were placed in a stereotaxic frame and an incision was made centrally along the scalp. A craniotomy positioned directly above the posterior BLA (stereotaxic coordinates in mm were AP: 3.1 ML: 4.5 and DV:7.8) was made bilaterally to allow microinjection needles to be lowered into the posterior BLA (BLA). Microinjection needles were used to transfect the BLA with 0.8–0.9 μL of a virus construct (pAAV5-CaMKIIa-hChR2(H134R)-EYFP) expressing the excitatory opsin, channelrhodopsin (ChR2) at a rate of 2 μL/min (Addgene, Cambridge, MA). Microinjection needles were maintained in place for 5 min before being withdrawn. Animals were sutured and allowed to recover in their home cages. The viral construct was allowed to express and traffic to BLA terminals for the following 5–6 weeks weeks and subsequently exposed to 10 days of chronic intermittent ethanol vapor inhalation or air exposure.

2.3 Chronic intermittent ethanol vapor inhalation exposure

All animals (Air control and CIE) were housed in their standard home cages on a reverse light cycle (9 p.m.–9 a.m.). Home cage housed animals in the CIE condition were placed in custom-built Plexiglas chambers (Triad Plastics, Winston-Salem, NC) to allow ethanol vapor to be pumped into the chamber for 12 h a day over the course of 10 days. Animals were weighed daily and tail blood samples were taken a minimum of 3 times during the 10 day CIE paradigm. Ethanol concentrations were adjusted maintain the animal's blood ethanol concentrations (BECs) at 150–225 mg/dL. Animals in the Air condition were exposed only to room air and handled daily to mimic handling of ethanol exposed animals. Following the 10 day procedure, ethanol exposed rats underwent 24 h of withdrawal (no ethanol vapor) before being sacrificed for electrophysiological recordings.

2.4 Blood ethanol concentrations

Blood ethanol concentrations (BECs) were determined from a 10 μL sample of tail vein blood obtained via tail snip from each individual rat. BECs were determined using a commercially available alcohol dehydrogenase enzymatic assay kit (Carolina Liquid Chemistries Corporation, Brea, CA). Ethanol concentrations were then determined using a spectrophotometer (Molecular Devices Spectra Max). The average BEC of all included animals was 177.13 ± 7.9 mg/dL.

2.5 Electrophysiology

Electrophysiological experiments were conducted following 24 h of withdrawal from CIE. Animals were deeply anesthetized using isoflurane. Following decapitation, the brain was removed rapidly and suspended in ice-cold NMDG recovery solution containing in mM: 92 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2·2H2O, and 10 MgSO4·7H2O. NMDG was titrated to pH 7.4 with 17 mL ± 0.5 mL of 5 M hydrochloric acid (REFS). Transverse slices containing the vHC were cut at a thickness of 300 μm using a VT1000S Vibratome (Leica Microsystems). vHC slices were placed in a holding chamber containing NMDG recovery solution. Slices were allowed to recover for 35 min before being transferred to a chamber filled with artificial cerebral spinal fluid (aCSF) containing in mM: 125 NaCl, 1.25 NaH2PO4, 25 NaHCO3, 10 D-Glucose, 2.5 KCl, 1 MgCl2, and 2 CaCl2. NMDG recovery and aCSF holding solutions were oxygenated with 95% O2 and 5% and warmed to 32–34 °C. For recordings, a single brain slice was transferred to a chamber mounted on a fixed stage under an upright microscope (Scientifica, SliceScope Pro 2000 microscope), where it was superfused continuously with warmed oxygenated aCSF. Whole-cell voltage-clamp recordings were made in presumptive pyramidal neurons of the ventral subiculum (vSub) using recording pipettes pulled from borosilicate glass (open tip resistance of 6–9 MΩ; King Precision Glass Co., Claremont, CA). The pipette solution contained (in mM): 130–140 Cs-gluconate, 10 HEPES, 1 NaCl, 1 CaCl2, 3 CsOH, 5 EGTA, 2 Mg2+-ATP, 0.3 GTP-Na2 and 2 Qx-314. Intracellular Cs+ was used as the primary cation carrier in voltage-clamp recordings to block K+ currents, including postsynaptic GABAB receptor-mediated currents, in the recorded neuron. Neurons in the ventral subiculum (vSub) were targeted for recording under a 40× water-immersion objective (numerical aperture = 0.8) with infrared-differential interference contrast (IR-DIC) optics, as described previously. Electrophysiological signals were obtained using a Multiclamp 700 B amplifier (Molecular Devices, Union City, CA), low-pass filtered at 3 kHz, digitized at 10 kHz, and recorded onto a computer (Digidata 1440 A, Molecular Devices) using pClamp 11.0 software (Molecular Devices). Series resistance, measured from brief voltage steps applied through the recording pipette (5 mV, 5 ms), was <25 MΩ and was monitored periodically during the recording. Recordings were discarded if series resistance changed by > 20% over the course of the experiment. Each recorded neuron represents an individual data point (n); recordings were made from at least four rats for each experimental group. To selectively stimulate terminals originating from the BLA and synapsing onto vSub neurons, blue light (473 nm) was delivered though the objective using a CoolLED driver (pE-300ulta). For recordings of optically-evoked excitatory and inhibitory postsynaptic currents (oEPSCs and oIPSCs, respectively), cells were voltage clamped at − 70 mV (near the theoretical IPSC reversal potential) and at 0 mV (near the theoretical EPSC reversal potential), respectively. To establish maximal monosynaptic amplitudes and polysynaptic areas under the curve cells, were stimulated for 5 ms and at 34 mW/mm2 intensity with the cell centered over the light source. Optical stimulation was performed at 0.1 Hz to obtain at least 10 consecutive optically-evoked postsynaptic currents. To establish the excitatory and inhibitory ratio (E/I ratio), the total excitatory area was divided by the total inhibitory area when both could be obtained from the same neuron. A minimum of 10 amplitude samples were averaged to arrive at an average response amplitude of presumptive monosynaptic oEPSCs. oEPSCs. Paired-pulse ratios (PPrs) were determined by delivering paired optical stimulations at an interstimulus interval (ISI) of 50,100 and 250 ms. Stimulation duration and intensity for PPr recordings were adjusted to obtain monosynaptic responses. PPrs were obtained by taking the ratio of the second and first amplitude (Amp 2/Amp 1). For recordings isolating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated oEPSCs cells and to establish the amplitude of NMDA-receptor- mediated oEPSCs cells were voltage-clamped at −80 and +40 mV, respectively. To determine the AMPA/NMDA ratio, stimulation duration and intensities were adjusted to obtain a minimum of 10 oEPSCs. The maximum NMDA-mediated amplitude was determined by subtracting the area of AMPA mediated activity from the area of AMPA/NMDA-mixed component. The AMPA/NMDA ratio was calculated as the ratio between these isolated components.

2.6 Drug application

For a subset of experiments several pharmacological reagents were bath applied. Picrotoxin was bath applied to block GABAA receptors to pharmacologically isolate oEPSCs for AMPA receptor-mediated oEPSCs and AMPA/NMDA ratios. Recordings in the presence of Tetrodotoxin (TTX; 1–2 μM; Tocris Bioscience, Minneapolis, MN) were made to record action potential-independent optically evoked activity. 4-Aminopyradine (500 μM) was added in combination with TTX to unmask optically evoked activity blocked by TTX. Picrotoxin (100 μM; Sigma-Aldrich, St. Louis, MO) was added to the ACSF to block GABAA receptors. For specific experiments, DL-2-Amino-5-phosphonopentanoic acid (AP-5; 100 μM), NMDA (300 μM), and 6,7-dinitroquinoxaline-2,3-dione (DNQX; 20 μM; all from Sigma-Aldrich) was added to ACSF to block AMPA receptor mediated conductance.

2.7 Statistical analysis

Statistical analysis was performed using MiniAnlaysis, Matlab and Prism. The normality of measures (amplitudes, areas, PPrs and ratios) was assessed using a Shapiro–Wilk test. Normally distributed data was analyzed using a two-tailed Student's T-Test, while non-normally distributed populations were compared using a Mann-Whitney Test. A repeated measures ANOVA was used to test the effect of multiple drug treatments of oEPSC. Significant differences between the means of Air and CIE measures were assessed using a two-tailed unpaired student's t-test or a Mann–Whitney test. All results were considered statistically significant with a p < 0.05. Errors are reported as ± SEM.

3 Results

One of the primary objectives of this study was to explore the role of CIE on synaptic transmission driven by BLA inputs to the vSub in female rats. To address this question, we virally transfected the BLA with a virus expressing the excitatory opsin, channelrhodopsin (ChR2). Subsequent to a trafficking window of 6 weeks, we targeted pyramidal neurons in the vSub for electrophysiological recordings while optically stimulating BLA fibers synapsing onto the recorded neuron using blue light (473 nm). We first measured optically evoked excitatory postsynaptic currents (oEPSCs) at −70 mV (near the IPSC reversal potential) (Smith et al., 2000). Inputs from the BLA elicited short latency oEPSCs (<5 ms) indicative of monosynaptic BLA inputs. In the majority of vSub neurons stimulation of BLA input led only to monosynaptic responses. However, in some cases, activation of this input led to recurrent excitation, likely driven by polysynaptic activity intrinsic to the vSub.

To assess the effect of CIE we first compared the amplitude of isolated (addition of TTX and 4 A P) monosynaptic oEPSCs (moEPSCs) of Air and CIE rats. We did not identify a change in monosynaptic BLA oEPSC amplitude (p = 0.5556, Air n = 9, CIE n = 13; Mann-Whitney Test; Fig. 1B and C) or oEPSC area (p = 0.7938, Air n = 9, CIE n = 13; Mann-Whitney Test; data not illustrated graphically). To address whether overall BLA-driven activity (mono- and polysynaptic) was impacted by CIE treatment we measured total charge transfer for the duration of the optically evoked BLA current in rACSF (in the absence of inhibitors). Here, we identified an increase in polysynaptic oEPSC area of CIE-treated rats (p = 0.0098; Air n = 26, CIE n = 24; Mann-Whitney Test; Fig. 1D and G). Activation of BLA inputs also elicited polysynaptic inhibitory currents (oIPSC) to vSub neurons. In response to CIE we saw an increase in the area of these oIPSCs (p = 0.0269; Air n = 27, CIE n = 23; Mann-Whitney Test; Fig. 1E and G). By recording both excitatory and inhibitory inputs in the same neuron from a subset of cells we were able to compare the excitatory to inhibitory balance (E-I ratio) in response to CIE. CIE animals did not show a shift in their E-I ratio (p = 0.665; Air n = 25, CIE n = 22; Mann-Whitney Test; Fig. 1F and G).Fig. 1 Poly-, but not monosynaptic, excitatory BLA inputs to the vSub are strengthened in response to CIE without impacting the E-I balance. A) Schematic illustration of recording configuration. Blue highlighted area signifies optical stimulation. B) Quantitative comparison of moEPSC amplitudes in Air and CIE rats. C) Representative moEPSC traces in the presence of TTX and 4 A P from an Air (top traces) and CIE (bottom traces) rat. In this and all other representative traces individual inputs are shown by thin gray lines while the average response amplitude is illustrated by a thicker overlaid black line. D-F) Quantitative comparison of oEPSC (D), oIPSC areas (E) and E/I area ratios (F). G) Representative traces of oEPSCs (upward deflecting traces) and oIPSCs (downward deflecting traces) obtained from Air (left) and CIE (right) rats. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

In addition to these measures, we also assessed possible presynaptic adaptations using optically driven paired-pulse ratios (oPPrs) in the pBLA-vSub pathway. We measured PPrs of oEPSCs with interstimulus intervals (ISI) of 50, 100 and 250 ms. We identified no differences in PPrs at 50 ms ISI (p = 0.4299; Air n = 5, CIE n = 5; unpaired t-test; Fig. 2A and B). At 100 ms (p = 0.0395; Air n = 17, CIE n = 9; t-test; Fig. 2C and D) and 250 ms (p = 0.0299; Air n = 13, CIE n = 9; unpaired t-test; Fig. 2E and F) ISI, on the other hand, we found a decrease in the PPr of CIE-treated between Air and CIE rats suggestive of a CIE-dependent increase in the release probability of pBLA-vSub inputs.Fig. 2 Presynaptic release probability of BLA input onto vSub neurons is increased in CIE rats. A) Quantitative graphical comparison of oEPSC PPrs at 50 ms ISI show no change in release probability between Air and CIE-treated rats B) Representative traces of oEPSC PPrs at 50 ms ISI. C and E) Quantitative graphical comparison of oEPSC PPrs at 100 (C) and 250 (E) ms ISI show an increase in release probability (decreased PPr) of CIE-treated rats. D and F) Representative traces of oEPSC PPrs at 100 (D) and 250 (F) ms ISI.

Fig. 2

The difference in CIE-associated adaptations between mono- and polysynaptic pBLA-vSub inputs may have been driven, at least in part, by changes in intrinsic vSub network activity. To address this possibility, we recorded both spontaneous EPSCs and IPSCs (sEPSCs and sIPSCs, respectively) from vSub pyramidal neurons of AIR and CIE-treated animals. We found no change, albeit a robust trend of an increase, in sEPSC frequency (p = 0.051; Air n = 18, CIE n = 18; Mann-Whitney Test; Fig. 3A and B) but did observe a significant increase in sIPSC frequency (p = 0.0086; Air n = 17, CIE n = 17; unpaired t-test; Fig. 3C and D). In contrast, CIE had no effect on the amplitude of either sEPSCs (p = 0.6240; Air n = 17, CIE n = 18; Mann-Whitney Test; Fig. 3A and B) or sIPSCs (p = 0.0987; Air n = 17, CIE n = 17; Mann-Whitney Test; Fig. 3C and D). These findings indicate that, in female rats, CIE primarily increased the presynaptic release probability of inhibitory inputs without impacting postsynaptic spontaneous neurotransmission properties.Fig. 3 Spontaneous inhibitory, but not excitatory, activity (sPSCs) is increased in CIE-treated rats. A) There is no CIE-dependent effect on sEPSC frequency (left graph) or amplitude (right graph). B) Representative sEPSC traces recorded from vSub neurons of Air (top two traces) and CIE (lower two traces) rats. C) Representative sIPSC traces recorded from vSub neurons of Air (top two traces) and CIE (lower two traces) rats. D) CIE elicits an increase in sIPSC frequency (left graph) but not amplitude (right graph). Blue line above upper traces indicate zoomed in section illustrated in lower traces. *p < 0.05 and errors are reported as ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The CIE-mediated increase in both pBLA-driven GABAergic inputs and intrinsic vSub GABAergic transmission raised our interest in exploring whether blocking GABAA receptor-mediated inhibition would impact optically driven and spontaneous activity in the vSub. To explore this question, we conducted electrophysiological recordings in the presence of the GABAA receptor antagonist picrotoxin (PTX). To focus our investigation on AMPA receptor-mediated adaptations, we conducted our electrophysiological recordings at −80 mV (where non-GluN3 containing NMDA receptors remain under voltage-dependent block (Mayer et al., 1984)) in the presence of PTX, while optically stimulating inputs arising from the BLA. Under these conditions we saw a robust increase in presumptive AMPA receptor-mediated excitatory polysynaptic vSub input in CIE rats (p = 0.0087; Air n = 19, CIE n = 13; Mann-Whitney Test; Fig. 4A and B). Additionally, we explored the effect of PTX on spontaneous activity by recording sEPSCs in the presence of PTX at −80 mV in vSub neurons. In the presence of PTX, CIE led to a robust increase in the frequency (p < 0.0001; Air n = 19, CIE n = 13; Mann-Whitney Test; Fig. 5A and B) of sEPSCs without a change in sEPSC amplitude (p > 0.9999; Air n = 19, CIE n = 13; Mann-Whitney Test; Fig. 5A and B) indicative of a presynaptic change in release probability that is unmasked by GABAA receptor-mediated disinhibition.Fig. 4 Blocking GABAA -mediated inhibition in the vSub shows a robust strengthening of BLA-driven inputs of vSub neurons from CIE-treated rats. A) Quantitative comparison of disinhibited (presence of PTX) BLA-driven inputs in Air and CIE-treated rats. B) Representative recordings of oEPSCs at −80 mV in the presence of PTX. **p < 0.01 and errors are reported as ±SEM.

Fig. 4

Fig. 5 Blocking GABAA -mediated inhibition in the vSub unmasks an increase in sEPSC activity. A) Representative recordings of oEPSCs at −80 mV in the presence of PTX. B) Quantitative comparison of disinhibited (presence of PTX) sEPSC frequency (top) and amplitude (bottom) in Air and CIE-treated rats. Blue line above upper traces indicate zoomed in section illustrated in lower traces. H ***p < 0.001 and errors are reported as ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Finally, to further assess AMPA receptor-mediated inputs we conducted recordings of BLA-vSub oEPSCs while voltage clamping vSub neurons at positive potentials (+40 mV) in the presence of PTX to unmask NMDA receptor-mediated inputs. With our prior data indicating a lack of effect of CIE on monosynaptic BLA-vSub excitatory neurotransmission, our principal analysis was not limited to putative monosynaptic vSub oEPSCs. Subsequent to recording a baseline mixed AMPA/NMDA-receptor component we isolated the AMPA receptor-mediated current by applying the NMDA receptor antagonist, AP-5. This allowed us to determine the magnitude of the current contributed by each receptor and consequently the AMPA/NMDA ratio. This approach revealed a significant CIE-dependent increase in the AMPA/NMDA ratio (p = 0.0047; Air n = 8, CIE n = 6; unpaired t-test; Fig. 6A and B). When possible, we did adjust stimulation parameters to capture putative monosynaptic BLA inputs. This analysis of moEPSCs also revealed an increase in the AMPA/NMDA ratio (p = 0.029; Air n = 6, CIE n = 4; unpaired t-test; data not shown).Fig. 6 AMPA/NMDA ratios are increased in vSub neurons of CIE rats. A) Quantitative comparison between Air and CIE rats show a strengthening of AMPA-mediated of BLA-driven inputs to vSub neurons in response to CIE. B) Representative recordings of oEPSCs at +40 mV in the presence of PTX before and after the application of the NMDA receptor antagonist AP-5. **p < 0.01 and errors are reported as ±SEM.

Fig. 6

4 Discussion

Increased synaptic excitation in the basolateral amygdala (BLA) and ventral hippocampus (vHC), as well as within a monosynaptic BLA-vHC circuit, are known to promote anxiety-like behaviors (Bach et al., 2023; Chang and Gean, 2019; Chang et al., 2015; Felix-Ortiz et al., 2013; Pi et al., 2020; Rau et al., 2015). Prior work by us and others also suggests that a strengthening of this circuitry may contribute to anxiogenic behaviors that develop during withdrawal from chronic alcohol vapor exposure (CIE) (Bach et al., 2021; Han et al., 2017; Morales et al., 2018; Price and McCool, 2022). We previously found that a 10 day CIE treatment followed by 24 h withdrawal increased anxiety-like behaviors in male and female rats, although these effects were more pronounced in males. Surprisingly, while extracellular vHC recordings taken at this same withdrawal timepoint revealed increased excitability in recordings from males, a decrease in vHC synaptic excitation was observed in females (Bach et al., 2021; Ewin et al., 2019; Pi et al., 2020). We recently followed up on these observations in males and also investigated the effects of CIE on the BLA projection to the vHC that is known to play a role in anxiety-like behaviors. As noted earlier, we discovered that vHC projecting BLA neurons innervate the ventral subiculum (vSub) and found that CIE led to increases in postsynaptic measures of synaptic excitability in the BLA-vSub circuit (e.g. AMPA/NMDA ratio, E/I ratio) as well as a presynaptic increase in spontaneous excitatory, but not inhibitory, intrinsic vSub synaptic transmission (Bach et al., 2021b). The present experiments, conducted in females, were designed to replicate the methodology of this prior study examining the effects of CIE on BLA-vSub transmission and intrinsic vSub synaptic activity to gain mechanistic insight into the sexually dimorphic effects of CIE that we had previously observed.

Our findings revealed that the BLA also strongly innervates the vSub of female rats. However, CIE had no effect on monosynaptic excitatory neurotransmission in the BLA-vSub pathway in females. When looking more broadly at BLA-vSub inputs composed of both mono- and polysynaptically-driven inputs, female rats did show an increase in both excitatory and inhibitory neurotransmission, similar to what we observed in males. However, unlike males, these adaptations were not associated with significant changes in E-I balance. We also discovered that CIE had sexually dimorphic effects on spontaneous vSub synaptic transmission, in that it led to a robust increase in spontaneous IPSC frequency in females, a finding we did not observe in males. Importantly, blocking GABAA receptors unmasked a significant CIE-associated increase in both BLA-vSub excitability and sEPSC frequency in female rats.

Overall, these findings suggest that intrinsic synaptic activity in the vSub is a principal driver of CIE-mediated adaptations and that this treatment has sexually dimorphic effects on vSub synaptic plasticity. While spontaneous inhibitory, but not excitatory, activity was increased in female rats, the opposite was true in males. Since inhibition of GABAergic transmission elicited a CIE-mediated increase in sEPSC frequency in both sexes, these data suggest that the increase in intrinsic inhibition in females prevented the CIE-associated intrinsic hyperexcitability observed in males. Notably, we also found that putative monosynaptic AMPAR-mediated postsynaptic conductance (obligatory use of GABAergic inhibition) was not increased in CIE-treated females, suggesting that intrinsic and/or BLA-vSub network-mediated GABAergic inhibition establishes an inhibitory shunt sufficient to block hyperexcitability in this circuit in female rats. Finally, CIE also increased the AMPA/NMDA ratio of putative monosynaptic responses in the presence of a GABAA-R blocker, but this treatment had no effect on putative BLA driven monosynaptic oEPSCs in the absence of a GABAAR inhibitor. A likely explanation for this finding is that CIE may have strengthened a recently discovered monosynaptic GABAergic projection from the BLA to the vSub (AlSubaie et al., 2021). Our preliminary data indicates that this projection is sufficient to elicit a significant reduction in the strength of monosynaptic glutamatergic input, arguing that this GABAergic conductance plays an important role in modulating monosynaptic BLA-vSub communication. Future studies are underway to explore the role of this novel BLA-vSub GABAergic projection in our CIE model.

The sex-dependent inhibitory adaptations are both intriguing as well as somewhat counterintuitive juxtaposed to adaptations of neuronal activity typically seen in preclinical models of anxiety, AUD and the available evidence in clinical studies (Bach et al., 2021b; Blume et al., 2019; Hernandez-Avila et al., 2004; Lack et al., 2007; Randall et al., 1999; Rau et al., 2015; Towers et al., 2023). In preclinical anxiety models, increases in BLA and vHC activity as well as BLA-vHC excitation has been associated with heightened levels of anxiety-like behavior (Bach et al., 2023; Felix-Ortiz et al., 2013; Rau et al., 2015; Silberman et al., 2009). Moreover, the frequent comorbidity that exists between anxiety disorders and AUD would lead one to predict that CIE increases excitability in these brain regions and circuit; a relationship we found to hold true in our prior studies in male rats (Bach et al., 2021b; Ewin et al., 2019). Prior behavioral evidence with CIE-treated female rats has shown more mixed findings, suggesting that female rats exposed to a ‘traditional’ 10-day CIE paradigm may be less susceptible to developing anxiety-like behaviors (Bach et al., 2021a; Ewin et al., 2019; Lack et al., 2007; Morales et al., 2018). Our current functional data indicating that CIE actually enhances BLA-vSub and intrinisic vSub GABAergic inhibition, supports the idea that female rats may be less susceptible to CIE and that sexually dimorphic adaptations in vSub inhibition may play an important mechanistic role in explaining these behavioral findings. Given that CIE is generally considered a model of alcohol dependence, this finding seems counterintuitive relative to most epidemiological data. In clinical populations, AUD remains more common in men, but heavy drinking women often experience more profound negative consequences and a heightened propensity to transition to AUD and associated comorbid conditions (Guinle and Sinha, 2020; Hernandez-Avila et al., 2004; Towers et al., 2023).

As in many such comparative analyses between clinical and preclinical studies, direct comparisons are plagued by a plethora of confounding factors. One such factor is that preclinical AUD models generally introduce alcohol to experimental subjects during adulthood. In humans, alcohol use is commonly initiated during adolescence or early adulthood and early onset alcohol exposure is strongly linked with increased risk of AUD (Jones et al., 2018). Adolescence and early adulthood are also periods of rapid hormone level changes that can profoundly influence synaptic plasticity (see additional discussion below). Indeed, there is emerging evidence from rodent and clinical studies that adolescence may be a particularly vulnerable period to experience excessive (or even moderate) alcohol exposure (Chassin et al., 2002; Creswell et al., 2020; Guinle and Sinha, 2020; McCool and McGinnis, 2020; Sicher et al., 2023). Thus, it will be important to consider how the sex differences in CIE-dependent synaptic adaptations that we have observed are influenced by CIE exposure during adolescence. Another important factor may be the duration of alcohol exposure used in preclinical CIE models. The normal progression of AUD typically involves the gradual development of pathologic levels of alcohol consumption over the course of months or years and the neural adaptations that develop in men and women during the first weeks of alcohol drinking are not known. These factors raise the question of whether the increase in vSub GABAergic inhibition that we observed in females following a relatively brief 10 day CIE treatment would persist with longer exposure durations. If this adaptation is transient, it is possible that longer more clinically-relevant CIE exposures could elicit similar maladaptive hyperexcitability in both sexes. Indeed, an early study found that a nine day chronic alcohol liquid diet significantly increased sensitivity to withdrawal-induced seizures triggered by the GABAA receptor antagonist bicuculline in male, but not female rats whereas after 14 days on the liquid diet, bicuculline withdrawal seizures thresholds were similar in both sexes (Devaud and Chadda, 2001). Moreover, a more recent study exploring CIE-dependent plasticity within the BLA of male and female rats found that females develop adaptations mimicking those of their male counterparts but at a slower progression. Notably, this study determined that presynaptic plasticity precedes postsynaptic adaptations (Morales et al., 2018). Our data identifying only pre-, but not postsynaptic, adaptations in the BLA-vSub pathway in female rats when we identified the opposite in male rats (post, but not presynaptic changes) would thus be congruent with the overall hypothesis that females may initially be protected from CIE-associated hyperexcitability through an elevation in GABAergic inhibition that may be overcome by more prolonged CIE exposures. Indeed, our preliminary data suggest that while a 10 day CIE treatment had no effect on anxiety measures in the elevated plus-maze, withdrawal from a longer 30 day exposure did significantly increase anxiety-like behavior on this assay.

Funding

This work was supported by the National Institutes of Health [grant numbers: K01 AA030081 (ECB), P50 AA06117 (JLW), R01 AA26551 (JLW), R37 AA017531 (JLW).

CRediT authorship contribution statement

Eva C. Bach: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Jeff L. Weiner: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Eva Bach reports financial support was provided by National Institutes of Health. Jeff L. Weiner reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.
==== Refs
References

Almonte A.G. Ewin S.E. Mauterer M.I. Morgan J.W. Carter E.S. Weiner J.L. Enhanced ventral hippocampal synaptic transmission and impaired synaptic plasticity in a rodent model of alcohol addiction vulnerability Sci. Rep. 7 1 2017 12300 10.1038/s41598-017-12531-z
AlSubaie R. Wee R.W. Ritoux A. Mishchanchuk K. Passlack J. Regester D. MacAskill A.F. Control of parallel hippocampal output pathways by amygdalar long-range inhibition Elife 10 2021 10.7554/eLife.74758
Bach E.C. Ewin S.E. Baldassaro A.D. Carlson H.N. Weiner J.L. Chronic intermittent ethanol promotes ventral subiculum hyperexcitability via increases in extrinsic basolateral amygdala input and local network activity Sci. Rep. 11 1 2021 8749 10.1038/s41598-021-87899-0 33888757
Bach E.C. Ewin S.E. Heaney C.F. Carlson H.N. Ortelli O.A. Almonte A.G. …Weiner J.L. Chemogenetic inhibition of a monosynaptic projection from the basolateral amygdala to the ventral hippocampus selectively reduces appetitive, but not consummatory, alcohol drinking-related behaviours Eur. J. Neurosci. 57 8 2023 1241 1259 10.1111/ejn.15944 36840503
Bach E.C. Morgan J.W. Ewin S.E. Barth S.H. Raab-Graham K.F. Weiner J.L. Chronic ethanol exposures leads to a negative affective state in female rats that is accompanied by a paradoxical decrease in ventral Hippocampus excitability Front. Neurosci. 15 2021 669075 10.3389/fnins.2021.669075
Blaine S.K. Sinha R. Alcohol, stress, and glucocorticoids: from risk to dependence and relapse in alcohol use disorders Neuropharmacology 122 2017 136 147 10.1016/j.neuropharm.2017.01.037 28159647
Blume S.R. Freedberg M. Vantrease J.E. Chan R. Padival M. Record M.J. …Rosenkranz J.A. Sex- and estrus-dependent differences in rat basolateral amygdala J. Neurosci. 37 44 2017 10567 10586 10.1523/JNEUROSCI.0758-17.2017 28954870
Blume S.R. Padival M. Urban J.H. Rosenkranz J.A. Disruptive effects of repeated stress on basolateral amygdala neurons and fear behavior across the estrous cycle in rats Sci. Rep. 9 1 2019 12292 10.1038/s41598-019-48683-3
Chang C.H. Gean P.W. The ventral Hippocampus controls stress-provoked impulsive aggression through the ventromedial hypothalamus in post-weaning social isolation mice Cell Rep. 28 5 2019 1195 1205 e1193 10.1016/j.celrep.2019.07.005 31365864
Chang C.H. Hsiao Y.H. Chen Y.W. Yu Y.J. Gean P.W. Social isolation-induced increase in NMDA receptors in the hippocampus exacerbates emotional dysregulation in mice Hippocampus 25 4 2015 474 485 10.1002/hipo.22384 25348768
Chappell A.M. Carter E. McCool B.A. Weiner J.L. Adolescent rearing conditions influence the relationship between initial anxiety-like behavior and ethanol drinking in male Long Evans rats Alcohol Clin. Exp. Res. 37 Suppl. 1 2013 E394 E403 10.1111/j.1530-0277.2012.01926.x Suppl 1 22924742
Chassin L. Pitts S.C. Prost J. Binge drinking trajectories from adolescence to emerging adulthood in a high-risk sample: predictors and substance abuse outcomes J. Consult. Clin. Psychol. 70 1 2002 67 78 Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/11860058 11860058
Christian D.T. Alexander N.J. Diaz M.R. Robinson S. McCool B.A. Chronic intermittent ethanol and withdrawal differentially modulate basolateral amygdala AMPA-type glutamate receptor function and trafficking Neuropharmacology 62 7 2012 2430 2439 10.1016/j.neuropharm.2012.02.017 22387532
Creswell K.G. Chung T. Skrzynski C.J. Bachrach R.L. Jackson K.M. Clark D.B. Martin C.S. Drinking beyond the binge threshold in a clinical sample of adolescents Addiction 115 8 2020 1472 1481 10.1111/add.14979 31984600
Devaud L.L. Chadda R. Sex differences in rats in the development of and recovery from ethanol dependence assessed by changes in seizure susceptibility Alcohol Clin. Exp. Res. 25 11 2001 1689 1696 Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/11707644 11707644
Erol A. Karpyak V.M. Sex and gender-related differences in alcohol use and its consequences: contemporary knowledge and future research considerations Drug Alcohol Depend. 156 2015 1 13 10.1016/j.drugalcdep.2015.08.023 26371405
Ewin S.E. Morgan J.W. Niere F. McMullen N.P. Barth S.H. Almonte A.G. …Weiner J.L. Chronic intermittent ethanol exposure selectively increases synaptic excitability in the ventral domain of the rat Hippocampus Neuroscience 398 2019 144 157 10.1016/j.neuroscience.2018.11.028 30481568
Felix-Ortiz A.C. Beyeler A. Seo C. Leppla C.A. Wildes C.P. Tye K.M. BLA to vHPC inputs modulate anxiety-related behaviors Neuron 79 4 2013 658 664 10.1016/j.neuron.2013.06.016 23972595
Felix-Ortiz A.C. Burgos-Robles A. Bhagat N.D. Leppla C.A. Tye K.M. Bidirectional modulation of anxiety-related and social behaviors by amygdala projections to the medial prefrontal cortex Neuroscience 321 2016 197 209 10.1016/j.neuroscience.2015.07.041 26204817
Flores-Bonilla A. Richardson H.N. Sex differences in the neurobiology of alcohol use disorder Alcohol Res 40 2 2020 4 10.35946/arcr.v40.2.04
Giacometti L.L. Barker J.M. Sex differences in the glutamate system: implications for addiction Neurosci. Biobehav. Rev. 113 2020 157 168 10.1016/j.neubiorev.2020.03.010 32173404
Gilpin N.W. Weiner J.L. Neurobiology of comorbid post-traumatic stress disorder and alcohol-use disorder Gene Brain Behav. 16 1 2017 15 43 10.1111/gbb.12349
Grace S. Rossetti M.G. Allen N. Batalla A. Bellani M. Brambilla P. …Lorenzetti V. Sex differences in the neuroanatomy of alcohol dependence: hippocampus and amygdala subregions in a sample of 966 people from the ENIGMA Addiction Working Group Transl. Psychiatry 11 1 2021 156 10.1038/s41398-021-01204-1 33664226
Grant B.F. Chou S.P. Saha T.D. Pickering R.P. Kerridge B.T. Ruan W.J. …Hasin D.S. Prevalence of 12-month alcohol use, high-risk drinking, and DSM-IV alcohol use disorder in the United States, 2001-2002 to 2012-2013: results from the national epidemiologic survey on alcohol and related conditions JAMA Psychiatr. 74 9 2017 911 923 10.1001/jamapsychiatry.2017.2161
Guinle M.I.B. Sinha R. The role of stress, trauma, and negative affect in alcohol misuse and alcohol use disorder in women Alcohol Res 40 2 2020 5 10.35946/arcr.v40.2.05
Han B.H. Moore A.A. Sherman S. Keyes K.M. Palamar J.J. Demographic trends of binge alcohol use and alcohol use disorders among older adults in the United States, 2005-2014 Drug Alcohol Depend. 170 2017 198 207 10.1016/j.drugalcdep.2016.11.003 27979428
Hernandez-Avila C.A. Rounsaville B.J. Kranzler H.R. Opioid-, cannabis- and alcohol-dependent women show more rapid progression to substance abuse treatment Drug Alcohol Depend. 74 3 2004 265 272 10.1016/j.drugalcdep.2004.02.001 15194204
Jones S.A. Lueras J.M. Nagel B.J. Effects of binge drinking on the developing brain Alcohol Res 39 1 2018 87 96 Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/30557151 30557151
Kim S.Y. Adhikari A. Lee S.Y. Marshel J.H. Kim C.K. Mallory C.S. …Deisseroth K. Diverging neural pathways assemble a behavioural state from separable features in anxiety Nature 496 7444 2013 219 223 10.1038/nature12018 23515158
Koob G.F. Volkow N.D. Neurobiology of addiction: a neurocircuitry analysis Lancet Psychiatr. 3 8 2016 760 773 10.1016/S2215-0366(16)00104-8
Lack A.K. Diaz M.R. Chappell A. DuBois D.W. McCool B.A. Chronic ethanol and withdrawal differentially modulate pre- and postsynaptic function at glutamatergic synapses in rat basolateral amygdala J. Neurophysiol. 98 6 2007 3185 3196 10.1152/jn.00189.2007 17898152
Mayer M.L. Westbrook G.L. Guthrie P.B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones Nature 309 5965 1984 261 263 10.1038/309261a0 6325946
McCool B.A. McGinnis M.M. Adolescent vulnerability to alcohol use disorder: neurophysiological mechanisms from preclinical studies Handb. Exp. Pharmacol. 258 2020 421 442 10.1007/164_2019_296 31595414
Morales M. McGinnis M.M. Robinson S.L. Chappell A.M. McCool B.A. Chronic intermittent ethanol exposure modulation of glutamatergic neurotransmission in rat lateral/basolateral amygdala is duration-, input-, and sex-dependent Neuroscience 371 2018 277 287 10.1016/j.neuroscience.2017.12.005 29237566
Pi G. Gao D. Wu D. Wang Y. Lei H. Zeng W. …Wang J.Z. Posterior basolateral amygdala to ventral hippocampal CA1 drives approach behaviour to exert an anxiolytic effect Nat. Commun. 11 1 2020 183 10.1038/s41467-019-13919-3 31924799
Price M.E. McCool B.A. Chronic alcohol dysregulates glutamatergic function in the basolateral amygdala in a projection-and sex-specific manner Front. Cell. Neurosci. 16 2022 857550 10.3389/fncel.2022.857550
Randall C.L. Roberts J.S. Del Boca F.K. Carroll K.M. Connors G.J. Mattson M.E. Telescoping of landmark events associated with drinking: a gender comparison J. Stud. Alcohol 60 2 1999 252 260 10.15288/jsa.1999.60.252 10091964
Rau A.R. Chappell A.M. Butler T.R. Ariwodola O.J. Weiner J.L. Increased basolateral amygdala pyramidal cell excitability may contribute to the anxiogenic phenotype induced by chronic early-life stress J. Neurosci. 35 26 2015 9730 9740 10.1523/JNEUROSCI.0384-15.2015 26134655
Sicher A.R. Starnes W.D. Griffith K.R. Dao N.C. Smith G.C. Brockway D.F. Crowley N.A. Adolescent binge drinking leads to long-lasting changes in cortical microcircuits in mice Neuropharmacology 234 2023 109561 10.1016/j.neuropharm.2023.109561
Silberman Y. Bajo M. Chappell A.M. Christian D.T. Cruz M. Diaz M.R. …Weiner J.L. Neurobiological mechanisms contributing to alcohol-stress-anxiety interactions Alcohol 43 7 2009 509 519 10.1016/j.alcohol.2009.01.002 19913194
Smith A.J. Owens S. Forsythe I.D. Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive J. Physiol. 529 Pt 3 2000 681 698 10.1111/j.1469-7793.2000.00681.x Pt 3 11118498
Towers E.B. Williams I.L. Qillawala E.I. Rissman E.F. Lynch W.J. Sex/Gender differences in the time-course for the development of substance use disorder: a focus on the telescoping effect Pharmacol. Rev. 75 2 2023 217 249 10.1124/pharmrev.121.000361 36781217
Vantrease J.E. Avonts B. Padival M. DeJoseph M.R. Urban J.H. Rosenkranz J.A. Sex differences in the activity of basolateral amygdalar neurons that Project to the bed nucleus of the stria terminalis and their role in anticipatory anxiety J. Neurosci. 42 22 2022 4488 4504 10.1523/JNEUROSCI.1499-21.2022 35477901
White A. Castle I.J. Chen C.M. Shirley M. Roach D. Hingson R. Converging patterns of alcohol use and related outcomes among females and males in the United States, 2002 to 2012 Alcohol Clin. Exp. Res. 39 9 2015 1712 1726 10.1111/acer.12815 26331879
