Glutamatergic dysfunction of astrocytes in paraventricular nucleus of thalamus contributes to adult anxiety susceptibility in adolescent ethanol exposed mice

All experimental procedures received approval from the Augusta University Institutional Animal Care and Use Committee and were conducted in accordance with NIH guidelines. C57BL/6 J mouse line (Catalog no. 000664) and the GFAP^cre/+ line [Catalog no. 024098; B6.Cg-Tg(Gfap-cre)77.6Mvs/2 J] were acquired from Jackson Laboratory (Bar Harbor, ME). GLT1-flox mice (Catalog no. 026619; B6.Cg-Slc1a2^tm1.1Ncd/J) [40] were received from Drs. Doo-Sup Choi at Mayo Clinic and Niels Christian Danbolt at University of Oslo. Ai9-GLT1 mice for cre-dependent overexpression of GLT1 were received from Drs. Doo-Sup Choi at Mayo Clinic and Ho Lee at Korea Cancer Center. Mice were accommodated in standard Plexiglas cages. The colony room was regulated to a consistent temperature of 24 ± 1 °C and humidity of 60 ± 2%, with a 12-h light/dark cycle (lights on at 07:00 a.m.). Mice were provided ad libitum access to food and water. Since accumulative rodent studies showed that early adolescent intermittent ethanol (AIE) exposure induces anxiety-like behaviors in adulthood regardless of sex [41, 42], in this study, we used both male and female mice.

Mice were anesthetized with isoflurane (1.5% in oxygen) and positioned on the rotational digital stereotaxic apparatus (RWD Life Science). The skull was aligned with a dual-tilt position equalizer and holes were drilled in the skull at the designated stereotaxic coordinates. Viruses were infused to the posterior parts of the PVT (AP −1.6 mm, ML + 0.0 mm, DV −3.0 mm from bregma) at a rate of 100 nl/min for 3 minutes via a 34-gauge needle (Catalog no. NF34BV; World Precision Instruments) with a micro-syringe pump (World Precision Instruments). The injection needle was kept in place for an extra 10 min following the injection. We injected the AAVs at the following titers: AAV₅-CaMKIIa-GCaMP6s, 4.7 × 10¹² GC/ml (UNC Vector Core); AAV₅-CaMKIIa-hM4Di-mCherry, 2.4 × 10¹³ GC/ml (Addgene); AAV₅-GFAP-mCherry, 3.1 × 10¹² GC/ml (Vector Biolab); AAV₅-GFAP-mCherry-Cre, 4.3 × 10¹² GC/ml (UNC Vector Core). We administered buprenorphine sustained release (1.3 mg/kg, s.c.; CoVetrus) to mitigate postoperative pain.

Brain slices containing the PVT region were prepared for electrophysiological recordings, as described [43]. Briefly, mice were deeply anesthetized by isoflurane inhalation, after which the brain was rapidly extracted and immersed in ice-cold sucrose-based artificial cerebrospinal fluid (aCSF) containing the following (in mM): 87 NaCl, 75 sucrose, 2.5 KCl, 11.25 NaH₂PO₄, 0.5 CaCl₂, 7 MgCl₂, 25 NaHCO₃, 0.3 l-ascorbate, and 25 glucose, and oxygenated with 95% O₂/5% CO₂. Coronal brain slices (300–350 μm) were cut with a vibrating compresstome (VF-310-0Z, Precisionary Instruments), subsequently placed in a slice holding chamber and incubated for 30 min at 34 °C and kept for at least 1 h at room temperature (24–25 °C) in carbonated (95% O₂/5% CO₂) standard artificial cerebrospinal fluid (aCSF) containing the following (in mM): 126 NaCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 2.5 KCl, 25 NaHCO₃, and 11 glucose. Electrical signals were captured using an Axon 700B amplifier, a Digidata 1550B A/D converter, and Clampfit 11.0 software (Molecular Devices). Throughout the experiments, the bath was consistently perfused with warm (32 °C) carbonated aCSF at a rate of 2.0–2.5 ml/min. Patch pipettes had a resistance of 4–6 MΩ when filled with a solution containing (in mM): 140 Cs-methanesulfonate, 5 KCl, 2 MgCl₂, 10 HEPES, 2 MgATP and 0.2 Na₂GTP for voltage clamping. The pH was adjusted to 7.2 with Tris-base and the osmolality to 310 mOsmol/L with sucrose. Healthy cells were identified using a high magnification microscope (at 400×, Nikon FN1 Microscope, Melville, NY) based on their morphology (round, ovoid, and non-swelled plasma membrane). The spontaneous firing was recorded using the loose-patch cell-attached method.

The in vivo electrophyiological recordings were performed as described [43, 44]. Briefly, mice were anesthetized by urethane (1.5 g/kg, i.p.; Sigma–Aldrich) [43] and positioned on a stereotaxic frame (RWD Life Science). We continuously monitored respiration rate and pedal withdrawal response during recordings, while the body temperature was maintained using a small-animal feedback-controlled warming pad (Kent Scientific Corporation). Small burr holes on skulls were created to insert high-impedance 32-channel microelectrodes (H10b, Cambridge NeuroTech). The reference wire (Ag/AgCl, 0.03 inches in diameter, A-M systems) was positioned in the contralateral parietal cortex. Electrophysiological signals were digitized at 20 kHz and band-pass–filtered from 300 to 3000 Hz (Intan Technologies). We verified single-cell spiking during the recording by checking spiking shapes using spike scope and comparing event timings using spike filter display in the Intan RHX Data Acquisition Software (Intan Technologies). The data were analyzed with Clampfit (version 11.2, Molecular Devices) and a custom-written code in MATLAB (R2019a, The MathWorks).

We monitored cellular Ca²⁺ transients in real-time in vivo by fiber-photometry as described [15, 44]. Three weeks post-injection of the AAV encoding GCaMP6s in the PVT, we implanted a fiber optic cannulae into the PVT (AP −1.40 mm, ML + 0.0 mm, DV −2.8 mm from bregma). The fluorescence signals were captured at 30 frames per second using a multi-wavelength fiber-photometry system (Plexon Multi-Fiber Photometry System, Plexon, Dallas, Texas), which had a dichroic mirror and a lens link to a photomultiplier tube (PMT) to reflect beams from 465 to 410 nm LED wavelengths. We used 410 nm as an isosbestic control to correct for calcium-independent fluorescence, movement artifacts, and photobleaching, 465 nm for the excitation of GCaMP6s. The collected data were analyzed using customized Mat-Lab codes and MatLab-based photometry modular analysis program, pMAT [45]. The ΔF/F is generated by subtracting the fitted isosbestic control (410 nm) from the signal (465 nm) to eliminate movement or other common artifacts. Then, those calcium transients were normalized as a Z-score. The Z-scores of spatiotemporal calcium transients were then sorted and averaged by animal location information (center vs. periphery) within a single animal and reported as the average Z-score across animals [46, 47].

Dreadd ligand, JHU37160, was purchased from Hello Bio (J60; JHU37160 dihydrochloride, Cat No. HB6261, Princeton, NJ), noted for its superior blood-brain barrier penetrance and enhanced affinity, potency, and selectivity of DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) [48]. For the activation of DREADDs, we administered J60 [0.3 mg/kg, dissolved in the vehicle, 1× phosphate-buffered saline (PBS)] intraperitoneally 10 min prior to the experiments in mice. The concentration has been demonstrated to have no off-target effects in rodent behavioral tests [48–52].

All experiments were conducted using a Bruker Biospec 7.0 Tesla 30 cm horizontal bore scanner (Bruker Biospin MRI GmbH, Germany), featuring a BGA12SHP gradient system that generates pulse gradients of 660 mT/m along each of the three axes, equipped with AVANCE III HD electronics and interfaced to a Bruker Paravision 6.0.1 console. A 4-channel surface phase array coil (Rapid MRI International, Columbus OH) served as the receiver, while a Bruker 86 mm linear-volume coil as the transmitter. Throughout the recordings, the animal was maintained under 1–2% isoflurane anesthesia and respiratory rate and body temperature were consistently monitored. The total duration of the whole imaging experiment was approximately 1.5 h. For 1H MRS, adjustments of all first- and second-order shims within the voxel of interest were accomplished with the MAPSHIM procedure. The in vivo shimming procedure yielded a full width half maximum (FWHW) ranging from 7.8 to 10.9 Hz for the unsuppressed water peak in the spectroscopic voxel of the mouse brain. The water signal was attenuated by variable power radiofrequency (RF) pulses with optimized relaxation delays (VAPOR). Outer volume suppression (OVS) integrated with a point-resolved spectroscopy (PRESS) sequence was employed for signal acquisition, utilizing 2 × 2 × 2 mm³ voxel, with a TR/TE = 2500/20 ms, a spectral bandwidth of 4 kHz, 2048 data points, and 256 averages. Spectral data were acquired from the voxel encompassing the dorsal thalamic brain region including the PVT according to the mouse brain atlas. Metabolite quantification was conducted utilizing the LC-Model (version 6.3-1 L) alongside the spectral basis set provided by the vendor. The absolute concentrations of the following metabolites were assessed: aspartate, creatine, γ-aminobutyric acid (GABA), glutamine, glutamate, guanidinoacetate, glycerophosphorylcholine, lactate, myo-inositol, N-acetylaspartate, N-acetylaspartylglutamate, Phosphocreatine, phosphorylcholine, and taurine. Only those results with Cramér-Rao lower bounds (CRLB, % SD) ≤ 50% were included in the statistical analysis.

All home cages housing mice were transported to the behavior testing room a minimum of 2 h before the testing start. The anxiety-like behaviors and spatial working memory were evaluated, and all the behaviors were assessed only during the light phase between 12 p.m. and 4 p.m. (Supplementary Fig.). The tests were performed in the same order; the mice underwent open field test (OFT) and elevated plus maze test (EPM). In the case of additional evaluations in light-dark test (LDT) and Y-maze, the mice underwent LDT and Y-maze. The behavioral experiments were conducted every other day during the same period of the day, because the period of the day can also have an impact on the behavior. The order of the animals was randomized. All the tests were scored using video tracking software, Ethovison (Ethovision XT, Noldus). 1

Tissue containing the PVT was punched out from coronal slices (1 mm thick) and homogenized in ice-cold RIPA lysis buffer (Thermo Fisher) containing a protease inhibitor cocktail (Roche). Equal amounts of protein extracts were denatured and subjected to SDS-PAGE using 4–12% Bis-Tris gels and transferred to PVDF membranes (Thermo Fisher). PVDF membranes were blocked with tris-buffered saline containing 0.05% Tween 20 and 5% (w/v) non-fat dried milk. These membranes were incubated with anti-GLT1 antibody (1:2000, Guinea pig, Millipore Sigma Cat# AB1783, RRID:AB_90949), anti-GLAST antibody (1:2000, Rabbit, Alomone Labs Cat# AGC-021, RRID:AB_2039885), anti-GAPDH antibody (1:2000, Mouse, Sigma Aldrich Cat# MAB374, RRID:AB_2107445), and anti-xCT antibody (1:1000, Rabbit, ABclonal Cat# A2413, RRID:AB_2863004) overnight at 4 °C. After washing with TBST, the membranes were incubated for 1 h with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The proteins were visualized by ECL solution (Thermo Fisher) using the G:Box Chemiluminescent Imaging System (Syngene).

Brains were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and transferred to 30% sucrose (Sigma-Aldrich) in PBS at 4 °C for 72 h. Brains were then frozen in dry ice and sectioned at 50 µm using a microtome (Precisionary, Ashland, MA). Brain slices were stored at −20 °C in a cryoprotectant solution containing 30% sucrose (Sigma-Aldrich) and 30% ethylene glycol (Sigma-Aldrich) in PBS. Sections were incubated in 0.3% Triton X-100 (Sigma-Aldrich), 3% bovine serum albumin in PBS for 1 h, followed by incubation with the primary antibody in 3% bovine serum albumin overnight at 4 °C. GLT1 colocalization was examined using primary antibodies against GLT1 (Rabbit, 1:500, Abcam Cat# ab205248, RRID:AB_2924274) with either of S100β (Guinea Pig, 1:500, Synaptic systems Cat# 287004, RRID:AB_2620025) for astrocytes or NeuN (Guinea Pig, 1:500, Synaptic systems Cat# 266004, RRID:AB_2619988) for neurons, followed by corresponding secondary antibodies conjugated with Alexa Fluor 594 (Goat anti-Rabbit IgG, 1:500, Abcam Cat# ab150180, RRID:AB_2650602) and Alexa Fluor 488 (Goat anti-Guinea Pig IgG, 1:500, Abcam Cat# ab150185, RRID:AB_2736871). After three washes in PBS, the sections were mounted onto a microscope slide and cover-slipped with mounting medium (Abcam). Images were captured on an LSM 700 laser scanning confocal microscope (Carl Zeiss) using a 10× or 40× water-immersion lens or a Nikon AX confocal microscope with NSPARC (Nikon) using a 20× lens. The Fluorescent intensity of the images was quantified using Image J (Fiji [64 bit], Image J ver. 1.54p).

For assessing the glutamate levels in the brain tissue, including the PVT, a colorimetric glutamate assay kit was used (Abcam, Cat# ab83389). The optical density (OD) value of each well was measured using a Spectrophotometer (Multiskan FC Photometer, Thermo Fisher) at an absorption wavelength of 450 nm for each assay.

Data were analyzed by unpaired t-tests, one-way and two-way ANOVAs, and post-hoc tests as appropriate and indicated for each experiment. All data are represented as mean ± SEM using Prism 10.1 (GraphPad Software, San Diego, CA). The statistical significance was set at p < 0.05. Detailed statistical tests and data with exact p values are listed in Supplementary Table 1.

To characterize the animal behaviors and brain adaptation induced by the adolescent chronic intermittent alcohol (ethanol) exposure (AIE) in adulthood, we exposed the mice to ethanol or counterpart air (CON) based on chronic intermittent ethanol exposure procedure when the animals were four weeks old (Fig. 1). The procedure persisted for three weeks and ended before the animals escaped the adolescent period. After four weeks withdrawal from the last ethanol exposure, we subsequently evaluated the anxiety-like behaviors through open filed test (OFT), EPM, and LDT (Fig. 1b–g and Supplementary Figure 1). While the total distance traveled was similar in both groups (Fig. 1d; Unpaired t-test, t = 0.1698, df = 16, P = 0.8673, N_mice = 9/group), the time spent in the center area in the OFT was significantly shorter in the AIE group compared to that in the CON group (Fig. 1c; Unpaired t-test, t = 2.966, df = 16, p = 0.0091, N_mice = 9/group). In EPM test, AIE group spent less time than the CON group in the open-arms (Fig. 1f; Unpaired t-test, t = 2.454, df = 16, N_mice = 9/group). Likewise, AIE group spent less time than the CON group in the bright light illuminated section during the LDT (Supplementary Fig. 1b–d; Unpaired t-test, t = 2.173, df = 14, p = 0.0474, Nmice = 8/group). Compared to the CON group, the AIE group mice did not exhibit significant changes in short-term spatial working memory in the spontaneous alternation behavior test (SAB) at the 4 weeks withdrawal from AIE procedure (Supplementary Fig. 1e–g; Unpaired t-test, t = 1.02, df = 14, p = 0.3254, N_mice = 8/group), indicating that repeated alcohol exposure during adolescent period increased the susceptibility to anxiety-like behaviors in adult.

Next, we examined whether the AIE procedure induces adaptation of the neuronal activities in the PVT, an area that has been considered a hub of arousal and anxiety network [24, 25, 55], via electrophysiology and temporal calcium imaging with fiber-photometry. In the in vivo electrophysiology, the basal rate of spontaneous spiking was significantly higher in the PVT neurons of AIE mice than that of CON mice (Fig. 1h–j; Unpaired t-test, t = 2.738, df = 40, p = 0.0092, N_cell = 20-22 [5 mice/group]). To further clarify whether the AIE-induced anxiety-like behaviors are related to the neuronal activities in the PVT, we measured spatiotemporal cellular activities of the PVT neurons with GCaMP6s, a genetically encoded Ca²⁺ indicator, synched with the behaviors in the OFT (Fig. 1k–m, Supplementary Fig. 2). The results showed that, AIE mice had enhanced calcium transients in the center area compared to that of CON group (Fig. 1k–m, two-way ANOVA F(1,32) = 34.54, P < 0.0001 for Group, post-hoc Center:CON vs. Center:AIE: p = 0.0001), while the mice did not show any group differences in the calcium transients when they stayed in the peripheral area (Fig. 1m, post-hoc Periphery:CON vs. Periphery:AIE: p = 0.9996). These data suggest that the hyperactivity of PVT neurons is accompanied with AIE-induced anxiety-like behaviors.

To assess whether the GLT1 reduction affects metabolites in the brain area, including glutamate in vivo, we measured the metabolites non-invasively using MRS in the mice with a GFAP-driven conditional heterozygous (cHET) reduction of GLT1 in the astrocytes (GLT1^cHET, GFAP^cre/+:GLT1^fx/+; Fig. 3e–k and Supplementary Fig. 3). Conditional targeting was verified with GFAP-promoter driven GLT1 conditional knock-out mice (GLT1^cKO, GFAP^Cre/+:GLT1^fx/fx; Fig. 3e, f, one-way ANOVA, F(2,9) = 34.20, P < 0.0001, N_mice = 4/group). Heterozygous reduction in GLT1 is sufficient to attenuate the GLT1 levels (post-hoc WT vs. GLT1^cHET: p = 0.001) without significant changes in GLAST (Supplementary Fig. 3a–c, one-way ANOVA, F(2,9) = 0.3250, P = 0.7307, post-hoc WT vs. GLT1cHET: p = 0.9962) and xCT (Supplementary Fig. 3a, b, one-way ANOVA, F(2,9) = 3.492, P = 0.0754, post-hoc WT vs. GLT1^cHET: p = 0.3683) levels in the PVT, similar to the changes by AIE (Fig. 3b–d). This heterozygous reduction in GLT1 also excludes the lethal phenotype shown in GLT1 cKO and global GLT1 KO [64, 65]. We confirmed that the reduction of GLT1 was in the astrocytes (Fig. 3g, h, two-way ANOVA, F(1,28) = 65.41, P < 0.0001, post-hoc S100β[+]:WT vs. S100β[+]:GLT1cHET: p < 0.0001, N_mice = 8/group), not in the neurons (post-hoc NeuN[+]:WT vs. NeuN[+]:GLT1cHET: p > 0.9999) of the PVT. The glutamate level in the thalamic area, including the PVT, was significantly reduced without changes in GABA levels, which induces the enhanced balance between glutamate and GABA ratio (Fig. 3i–k, Unpaired t-test, t = 2.497, df = 18, p = 0.0225, N_mice = 10/group). When we measured their behaviors in the OFT, the time spent in the center was significantly reduced in the GLT1^cHET mice compared to that in the CON mice (Fig. 3i–m, Unpaired t-test, t = 2.700, df = 14, p = 0.0173, N_mice = 8/group), with no change in distance traveled (Fig. 3n, Unpaired t-test, t = 1.472, df = 14, p = 0.1632). In the EPM test, the GLT1^cHET mice also showed the reduced time spent in open-arms (Fig. 3o–p, Unpaired t-test, t = 2.591, df = 14, p = 0.0213, N_mice = 8/group), with no change in distance traveled (Fig. 3q, Unpaired t-test, t = 0.3342, df = 14, p = 0.7432), suggesting the GLT1 reduction in astrocytes increase the thalamic glutamate levels and induces anxiety-like behaviors.

Taken together, our findings support the hypothesis that adolescent repeated ethanol exposure alters the capacity for glutamatergic communication between astrocytes and neurons in the PVT, leading to the anxiogenic phenotypes in adult, at least partly, via the astrocyte glutamate transporter, GLT1.

Similar to the previous reports of the anxiogenic effects of ethanol on rodents [16, 17, 66–69], we described anxiety-like behaviors in mice withdrawn from repeated ethanol exposure during the adolescent period, as shown by the reduced time spent in the center of an open field, and in the open arms of an elevated plus maze, as well as the less time in the light illuminated area. The total distance traveled remained unchanged, indicating the adolescent repeated ethanol exposure did not significantly impact the locomotor activity in our model. Notably, we observed the adaptation of cellular activities in the PVT. Although the PVT has been receiving increasing attention to be linked with anxiety-related behaviors, especially behavioral malfunction induced by adverse experiences during early in life [20], its role in the context of withdrawal from adolescent repeated ethanol exposure was unknown. We found that PVT neurons in ethanol-withdrawn adults (AIE) had a significantly higher spontaneous firing rates, suggesting a possible contribution of PVT hyperactivity to the increased anxiety levels. This possibility was supported by our data showing that selective inhibition of PVT neurons by chemogenetic approach or transgenic upregulation of GLT1 in the PVT mitigated elevated anxiety-like phenotypes associated with the ethanol withdrawal. Searching for the underlying cellular and molecular mechanisms, we found that the PVT neurons of AIE mice had an enhanced level of glutamate and glutamate/GABA ratio, compared to CON mice and this is accompanied with the reduction of GLT1 levels in the PVT, suggesting that GLT1 dysregulation may contribute to the observed increased activities of PVT neurons in the AIE mice. These data support a functional relationship between anxiety-like behaviors and the activities of PVT. The PVT is one of the main upstream inputs to the amygdala, a region recognized as a central hub within anxiety-processing networks and previously reported to undergo adaptive changes after long-term withdrawal reaching to adulthood from AIE exposure [69]. Recent anatomical and functional studies, dissecting the roles of PVT in an anterior-posterior axis and amygdala in a medio-lateral axis, demonstrate that the posterior PVT (pPVT) maintains robust projections to the central amygdala (CeA) and plays a critical role in anxiety modulation [70]. Compared to the anterior part of PVT (aPVT), the pPVT exhibits greater involvement in pain-induced anxiety processing, which aligns with our findings regarding PVT hyperactivity in adapted anxiogenic states. Given emerging evidence for functional diversity along the anterior-posterior axis of the PVT, where distinct subregions differently modulate amygdala sub-regional populations and anxiety-related behaviors, future investigations should examine how PVT subregional astrocyte populations influence downstream amygdala circuitry and broader multiple regional anxiety-networks. Such studies would advance our understanding of how local events by astrocyte-neuron interactions contribute to whole-brain homeostatic regulation.

The observation of prolonged brain adaptation, including the reduction of GLT1 protein expression in AIE mice may be caused by changes in the factors that affect the corresponding gene expression. For example, the transcription factor, Pax6, has been identified as a common mechanism of regulating GLT1 expression, without expression on the levels of another astroglial glutamate transporter, GLAST, by binding within the upstream of the GLT1 translation start site [71], which is also aligned with our findings in the PVT. Indeed, the Pax6 is expressed early in development, predominantly in a few body parts, including the brain [72], and has an essential role for early animal development [73, 74]. While the expression of Pax6 is reduced after ethanol exposure [75], Pax6 overexpression rescued alcohol-induced cellular dysfunction [76]. In addition, multiple epigenetic mechanisms via microRNAs (miRNAs), which are short non-coding RNAs modulating mRNA translation, have been suggested to contribute to the brain adaptation due to alcohol exposure during adolescence, which increase the susceptibility in adulthood to alcohol use and anxiety [42, 66, 69, 77, 78]. Thus, given the role of miRNAs and epigenetic impacts in the regulation of transcription factors, including Pax6 [79–81], it is intriguing to investigate the mechanisms how the synergistic reciprocal epigenetic modulations with related transcription factors affect the selective changes in GLT1 of the PVT by the adolescent repeated ethanol.

Although we did not identify the cell type we recorded in the current study, it has been known that the neurons in the PVT are majorly glutamatergic [82–84]. Recently, accumulative observations have provided the additional information there are diverse neuronal subpopulations in the PVT according to significant transcriptomic variance corresponding to the anterior-posterior axis [85, 86], which may contribute to difference multi-brain regional connectivity and diverse behaviors. Importantly, not only Central and Basolateral Amygdala (CeA, BLA) by direct and indirect connection, PVT also has been well known to have a role of pivotal region for anxiety-network including the bed nucleus of the stria terminalis (BNST) and Nucleus Accumbens (NAc) [25], where are the largest cluster of anxiety-network in the rodent and primate brains, participating in mediation of generalized anxiety and related arousal behaviors [87, 88], and environment-raised anxiety and reward motivation behaviors [89, 90], respectively. However, the functional significance of the various sub-connections from the PVT during ethanol withdrawal, especially adapted by the repeated exposure to ethanol during adolescent period, remains poorly addressed. Thus, future studies are also needed to clarify which cell-types and circuits are more significantly impacted by adolescent repeated ethanol exposure in an age-dependent manner. In addition, given the role of PVT in arousal and vigilance-linked behaviors and the connection between anxiety and hyperarousal, it will be intriguing to investigate the role of PVT GLT1 in arousal-related behavioral patterns, including social behaviors [25, 27, 91] and sleep-wake cycles [92, 93]. One limitation of our study is the exclusion of sex differences by the use of both male and female mice. Although previous studies showed that adult anxiety induced by AIE is observed in a sex independent manner [41, 42], many of the psychiatric and neurological disorders induced by AIE are manifest only observed in one sex [13]. Thus, age- and sex- dependent evaluation need to be further studied as well.

Our data here suggests that GLT1 downregulation-induced PVT hyperactivity in adults exposed to adolescent repeated ethanol exposure contributes to anxiety-like behaviors. This study implies the importance of glutamatergic homeostatic balance through GLT1 in the adult behavioral susceptibility induced by the early life adverse experience.