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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Neuropharmacology. 2021 Dec 10;205:108918. doi: 10.1016/j.neuropharm.2021.108918

Astrocytes play a critical role in mediating the effect of acute ethanol on Central Amygdala glutamatergic transmission

Mariam Melkumyan 1, Angela E Snyder 1, Sarah S Bingaman 1, Amy C Arnold 1, Yuval Silberman 1
PMCID: PMC8792276  NIHMSID: NIHMS1765009  PMID: 34896402

Abstract

The Central Amygdala (CeA) has been heavily implicated in many aspects of alcohol use disorder. Ethanol (EtOH) has been shown to modulate glutamatergic transmission in the lateral subdivision of the CeA, however, the exact mechanism of this modulation is still unclear. EtOH exposure is associated with increased pro-inflammatory cytokines in the CeA, and inhibition of neuroimmune cells (microglia and astrocytes) has previously been shown to reduce EtOH drinking in animal models. Since neuroimmune activation seems to be involved in many of the effects of EtOH, we hypothesized that acute EtOH exposure will increase excitatory glutamatergic transmission in the CeA via modulation of neuroimmune cells. Using ex vivo brain slice whole-cell patch clamp electrophysiology, it was found that a physiologically relevant concentration of EtOH (20mM) significantly increased presynaptic glutamatergic transmission in the CeA. Pharmacologic and chemogenetic inhibition of astrocyte function significantly reduced the ability of EtOH to modulate CeA glutamatergic transmission with minimal impact of microglia inhibition. This finding prompted additional studies examining whether direct neuroimmune activation through lipopolysaccharide (LPS) might lead to an increase in the glutamatergic transmission in the CeA. It was found that LPS modulation of glutamatergic transmission was limited by microglia activation and required astrocyte signaling. Taken together these results support the hypothesis that acute EtOH enhances lateral CeA glutamatergic transmission through an astrocyte mediated mechanism.

Keywords: ethanol, glutamate, central amygdala, astrocytes, microglia, lipopolysaccharide, mouse, electrophysiology, DREADDs

Introduction

The Central Amygdala (CeA) is involved in processing emotional stimuli and is critically involved in alcohol use disorder (AUD) and substance use disorder (Gilpin and Roberto, 2012; Roberto et al., 2020). For example, lesions of the CeA blocked alcohol (EtOH) drinking in a two bottle choice paradigm (Möller et al., 1997), and chemogenetic manipulation of the CeA affected drinking behaviors in rats (Haaranen et al., 2020), suggesting that the CeA is critically involved EtOH intake (Koob and Volkow, 2010). Therefore, determining the mechanism(s) by which EtOH may modulate CeA neurocircuit function may uncover new AUD treatment targets. Previous research has focused on EtOH modulation of GABA signaling, predominantly in the medial subdivision of the CeA (Bajo et al., 2014, 2008; Cruz et al., 2011; Herman and Roberto, 2016; Roberto et al., 2004; Schweitzer et al., 2016). While studies mainly conducted in the medial subdivision of the CeA have shown that acute EtOH administration either increases or decreases glutamatergic transmission (Gilpin and Roberto, 2012; Herman et al., 2016; Roberto, 2004; Roberto et al., 2012), other work indicates that EtOH can increase glutamatergic transmission in the CeA, at least in the lateral subdivision (Roberto et al., 2020; Silberman et al., 2015). Therefore, additional studies are necessary to fully elucidate the mechanism of EtOH action on CeA glutamatergic neurotransmission.

Neuroinflammation appears to be a key contributor to EtOH actions in the brain, and previous research indicates that neuroimmune signaling plays a key role in EtOH modulation of CeA GABAergic transmission. Chronic EtOH exposure and alcohol dependence have been shown to increase proinflammatory markers in the CeA (Patel et al., 2019), and inhibition of cytokines has been shown to reduce EtOH consumption (McCarthy et al., 2018; Truitt et al., 2016). Additionally, chemogenetic inhibition of astrocytes in the central and basolateral amygdala and pharmacologic inhibition of microglia has been shown to reduce drinking (Agrawal et al., 2011; Nwachukwu et al., 2021). Furthermore, in the CeA, neuroimmune activation increases GABAergic inhibitory postsynaptic potentials (Bajo et al., 2014) – a mechanism that potentiated the effects of acute EtOH. Additional studies by the same group showed that interleukin-1β (IL-1β) may alter the basal GABAergic transmission in the CeA and interact with the EtOH-induced facilitation of GABAergic transmission in the CeA (Bajo et al., 2015). Spontaneous GABAergic transmission was also shown to be affected by the interaction of toll-like-receptor-4 and corticotropin releasing factor mediating the relationship between EtOH and stress (Varodayan et al., 2018).

Questions still remain about the effect of neuroimmune cells and EtOH on the glutamatergic transmission in the CeA. Our previous work (Silberman et al., 2015) indicates that ethanol increases glutamatergic transmission, although the mechanisms are unclear. It is known that astrocytes are involved in the regulation of synaptic glutamate transmission and clearance (Murphy- Royal et al., 2017). Since neuroimmune cells seem to be involved in many of the effects of EtOH and act on the glutamatergic system, we hypothesized that acute EtOH exposure may increase excitatory glutamatergic neurotransmission in the CeA via modulation of neuroimmune cells. Utilizing both pharmacologic and chemogenetic inhibition of neuroimmune cell activity in ex vivo brain slice electrophysiology experiments, it was found that EtOH-induced increases in CeA glutamatergic transmission were mediated by astrocytes, with minimal microglia contribution.

Materials and Methods

Animals

84 adult C57Bl/6J (41 male, 43 female, The Jackson Labs, strain ID 000664) mice were used in all the studies. For electrophysiological experiments targeting the CeA, 54 mice (>7 weeks old) were used for lipopolysaccharide (LPS) (Sigma, cat. no. L4391) or EtOH (Pharmco, cat. no. 111000190) application in electrophysiology experiments. 19 of these mice were first utilized to deliver a GFAP-DREADD virus into the CeA prior to pharmacologic examination with electrophysiology. Prior to GFAP-DREADD electrophysiology studies, a group of 5 mice was used to assess and ensure proper viral delivery of GFAP DREADDs into the CeA microinjection site. A group of 6 mice (3 males and 3 females) were used for sulforhodamine 101 staining. All mice were housed in groups of two to five for the duration of the studies. Food and water were available ad libitum. All procedures were approved by the Institutional Animal Care and Use Committees at Penn State College of Medicine (Hershey, PA).

Microinjection Surgeries

For microinjection studies, the protocol outlined in Silberman et al., 2015 was followed. Specifically, mice >7 weeks of age were used for the viral injection studies. The mice were anesthetized using isoflurane, placed in the stereotaxic apparatus (Leica Angle Two) and injected intracranially with 300nl at 60nl/min of AAV5-GFAP-hM4D(Gi)-mCherry (Addgene, #50479) bilaterally into the CeA (AP: −1.6, ML: ±2.5, DV: −4.25) based on the coordinates from the Franklin and Paxinos, 2008 atlas. Mice were given 5mg/kg ketoprofen once every 24 hours for 48 hours post-surgery. The mice were euthanized 6–12 weeks after the injections for immunohistochemistry or electrophysiology studies.

Electrophysiology

Brain slice preparation:

Coronal brain slices of 250μm-thickness containing the CeA were prepared using a Leica VT1200s vibratome with a ceramic blade (Campden Instruments Limited, part # 7550–1-C) from adult C57Bl/6J mice described above. Mice were anesthetized with isoflurane and were perfused with 10mL ice-cold oxygenated sucrose dissecting solution (see below) through the left ventricle of the heart. The brains were dissected and submerged in the ice-cold, oxygenated sucrose dissecting solution (in mM: 183 sucrose, 20 NaCl, 0.5 KCl, 1 MgCl2, 1.4 NaH2PO4, 25 NaHCO3, and 10 glucose). Following dissection and cutting, the 250μm-thick coronal brain slices containing the CeA were placed in an oxygenated holding solution with a modified artificial cerebrospinal fluid (ACSF) (in mM: 100 sucrose, 60 NaCl, 2.5 KCl, 1.4 NaH2PO4, 1.1 CaCl2, 3.2 MgCl2, 1.2 MgSO4, 22 NaHCO3, 20 glucose, 1 ascorbic acid) for 14 minutes. After 14 minutes, the slices were transferred to an oxygenated standard ACSF solution (in mM: 124 NaCl, 4.4 KCl, 2CaCl2, 2.95 MgSO4, 1 NaH2PO4, 10 glucose, 26 NaHCO3) at 28–32°C for at least 30 minutes. For drug pretreatment studies, a slice was placed in an oxygenated standard ACSF and the appropriate drug at final concentration: 100μM minocycline (TCI, cat. no. M2288) was used as a microglia inhibitor, 100μM fluorocitrate (Sigma, cat. no. F9634) was used as an astrocyte inhibitor (Yang et al., 2019), and 10μM Clozapine-N-Oxide (CNO) (Tocris, cat. no. 6329) was used for the inhibitory DREADDs for at least 30 min prior to recordings. In the recording chamber, the slices were perfused with standard oxygenated ACSF (with appropriate pretreatment drugs at final concentration, if applicable) at a flow rate of 2mL/min at room temperature.

Whole-cell patch clamp recordings:

We targeted our recordings to the lateral subdivision of the CeA. Whole-cell patch clamp recordings of AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSC) were pharmacologically isolated by adding 25μM picrotoxin (PTX) (Tocris, cat. no. 1128) to standard ACSF and were recorded by holding the cell at −70mV. Recording electrodes were filled with a potassium gluconate (KGlu)-based internal solution consisting of: (135 mM K+-gluconate, 5mM NaCl, 2mM MgCl2, 10mM HEPES, 0.6mM EGTA, 4mM Na-ATP, 0.4mM Na-GTP, pH 7.34–7.36, 285–290 mOsmol). Following a stable baseline recording period of at least 6 minutes, 20mM EtOH or 500ng/mL LPS was bath-applied for 10 minutes followed by a 10-minute wash. Recordings were performed in 2-minute epochs in gap-free mode. For the antagonist studies, the appropriate drug (100μM minocycline or 100μM fluorocitrate) was bath-applied throughout the recording. For the inhibitory DREADDs studies, a standard concentration (Kayyal et al., 2019; Li et al., 2016) of 10μM CNO was bath-applied throughout the recording. Electrophysiology recordings were made using Sutterpatch7 and analyzed using Sutterpatch8.04. Summary of the drug effect was calculated using the peak change during a two-minute epoch following drug application (typically the last two to four minutes of drug application and/or first two minutes of washout period) normalized to the average of the 6 minutes of baseline. The time point for peak drug effects was chosen as there is variability in rate of ACSF perfusion from day to day but generally occur between 8–10 minutes of drug-on period and first 2 minutes of drug-off period. Cells with more than 20% change in access resistance or otherwise unstable cells (e.g., shifting holding currents during recordings), were removed from final data analysis.

Immunohistochemistry

For immunohistochemistry (IHC) the protocol outlined in Aimino et al., 2018 was followed with some modifications. AAV5-GFAP-hM4D(Gi)-mCherry microinjected mice not used for electrophysiology were anesthetized using isoflurane and perfused with 10 mL 0.1M phosphor-buffered saline (PBS, diluted from Sigma-Aldrich, cat. No. P3619) followed by 15 mL of 4% paraformaldehyde in 0.1M PBS. The brains were excised, post-fixed in 4% PFA/PBS overnight at 4°C, placed in 30% sucrose in PBS solution for 3 days at 4°C, and then stored in cryoprotectant prior to use as outlined in Aimino et al., 2018. Coronal slices of 40μm-thickness containing the CeA were cut on cryostat (Leica CM1850) and stored in cryoprotectant before fluorescent IHC staining. Free-floating slices containing the CeA were washed in PBS (4×10min), permeabilized with 0.5% Triton X-100 in PBS (30min) and blocked with 10% Normal Donkey Serum in PBS containing 0.1% Triton X-100 (1h). To label astrocytes, slices were incubated with Rabbit Anti-S100 antibody (1:500, Abcam, ab868) for 72 hours at 4°C. The slices were washed with PBS (4×10min) and then incubated with donkey anti-rabbit Alexa Fluor 488 (1:250, Jackson ImmunoResearch Laboratories, cat. no. 711–546-152) fluorescent dye-conjugated secondary antibody with 0.1% Triton X-100 in PBS for 24 hours. Following a 24-hour incubation, slices were washed, mounted, and cover-slipped using Prolong Gold Antifade Mountant with DAPI (Invitrogen, cat. no. P36931). The stained CeA slices were z-stack-imaged using Zeiss Axio Examiner.Z1 confocal microscope. The images were autocorrected for image brightness using Zen3.3 (blue edition) software.

Sulforhodamine 101 staining

250μM thick slices were prepared as outlined in the Electrophysiology - Brain slice preparation section above. The slices receiving fluorocitrate pretreatment, after at least 30 minutes of incubation in oxygenated ACSF, were transferred to oxygenated ACSF solution containing 100uM fluorocitrate to be incubated for 20 minutes. After the incubation, slices were moved to oxygenated ACSF containing 1μM Sulforhodamine 101 (SR101) (Sigma-Aldrich, cat. No. S7635) and 100μM fluorocitrate for 20 minutes to stain astrocytes for imaging. After the 20 minutes, the slices were incubated in ACSF containing 100μM fluorocitrate for 20 minutes, following which the slices were moved to 4% PFA in PBS overnight. The slices were then moved to PBS until imaging with the Zeiss confocal microscope. For vehicle slices, the oxygenated ACSF solution did not contain fluorocitrate in any of the steps. The slices were imaged using a 20x objective with 1.3x magnification with the final image having an area of 0.22mm2. The data was quantified using ImageJ to count the number of cells in the 0.22mm2 area in each slice. Two slices per animal were used, with the final data being an average of the number of astrocytes across the two slices per animal.

Statistical analyses

Statistical analysis was conducted using Microsoft Excel for Microsoft 365 MSO and GraphPad Prism 9. GraphPad Prism 9 and Microsoft PowerPoint for Microsoft 365 MSO were used for figure preparation. One sample t-test was used to determine whether EtOH and LPS significantly increase glutamatergic transmission compared to pre-drug baseline. To determine whether inhibitor pretreatments have an effect on EtOH or LPS modulation of glutamatergic activity, one-way ANOVA with Tukey’s multiple comparisons test or Student’s t-test was used to compare the peak drug effects, as applicable. For sex differences, a two-way ANOVA was used where applicable. For IHC, Zen 3.3 (blue edition) was used to stitch the images and adjust the color threshold. All values throughout the study are presented as mean±SEM.

Reagents

For electrophysiological experiments, all drugs were diluted in diH2O to make stock solutions, except PTX, which was diluted in dimethyl sulfoxide (DMSO) (final DMSO concentration of 0.02% vol/vol in ACSF). Stocks were then directly added to ACSF at final concentrations. All reagents were purchased from Sigma-Aldrich unless otherwise stated above.

Results

EtOH increases presynaptic glutamatergic transmission in lateral CeA through an astrocyte dependent mechanism

Previous work has shown that 1) EtOH increases glutamatergic transmission in the CeA (Silberman et al., 2015); 2) EtOH interacts with neuroimmune signaling to modulate CeA GABA transmission (Bajo et al., 2015, 2014; Patel et al., 2019); and 3) neuroimmune cells regulate glutamatergic transmission in the basolateral amygdala and other synapses in the central nervous system (Murphy- Royal et al., 2017; Nwachukwu et al., 2021; Yang et al., 2019). This suggests that neuroimmune cells, such as microglia and astrocytes, might play a role in the EtOH modulation of glutamatergic transmission seen in previous studies. Therefore, we hypothesized that inhibition of astrocytes and microglia will attenuate the EtOH induced increase in glutamatergic transmission in the CeA. We first sought to examine the effect of pharmacologically relevant concentrations of EtOH on glutamatergic transmission in the CeA. Whole-cell patch clamp electrophysiology studies showed that a 10-minute bath application of 20mM EtOH resulted in a significant increase in sEPSC frequency (61.36±14.49% increase from baseline, n=15, t=4.235, p=0.0008, fig. 1A, B, C) with no significant effect on sEPSC amplitude (−3.116±5.134% from baseline, n=15, t=0.6070, p=0.5536, fig. 1E). Peak effects of EtOH application typically coincided with the last two minutes of EtOH application and first two minutes of washout (fig 1B). Next, we pretreated slices with either fluorocitrate – an astrocyte inhibitor – or minocycline – a microglia inhibitor – to see whether these cells are involved in the mechanism of EtOH action on the CeA glutamatergic transmission. After a 30-minute pretreatment and continued bath application of fluorocitrate during recordings, a one-way ANOVA (F(2,39)=4.820, p=0.0135) with Tukey’s post-hoc comparison revealed that the ability of EtOH to increase sEPSC frequency was significantly attenuated by fluorocitrate (2.392±5.235% from baseline, n=13, p=0.0106 compared to EtOH alone, fig. 1B,C), suggesting that the EtOH induced increase of glutamatergic transmission in the CeA requires astrocyte signaling. To assure that fluorocitrate inhibits astrocyte function, the slices were treated with 1μM SR101 with or without 100μM fluorocitrate pretreatment. The results indicate significant reduction (t=3.241, p=0.0089) in SR101 labelled cells after fluorocitrate pretreatment (13.42±3.305 SR101 positive cells, n=6) compared to vehicle (35.17±5.840 SR101 positive cells, n=6) (fig. 1F, G). One-way ANOVA indicated that minocycline pretreatment did not significantly alter the ability of EtOH to increase sEPSC frequency; however, one-sample t-test showed that there was a trend toward a reduction in the magnitude of EtOH effects on sEPSC frequency compared to baseline under these conditions (26.30±16.88% from baseline, n=14, p=0.1432, fig. 1B, C). There were no sex differences in the effect of EtOH on sEPSC frequency between any of the conditions (F(1, 38)=0.1214, p=0.7294, fig. 1D), therefore, the combined data in fig. 1C was utilized for conclusions. Additionally, there was no significant effect of EtOH on sEPSC amplitude in any condition tested (F(2, 37)=2.666, p=0.0829, fig. 1E). Importantly, there were no significant differences in baseline sEPSC frequency or amplitude under any of the treatment conditions (Table 1). Overall, this data suggests that EtOH requires astrocytes activation to increase the glutamatergic transmission in the CeA through a presynaptic mechanism.

Figure 1: Ethanol increases presynaptic glutamatergic transmission in the CeA through astrocytic activation.

Figure 1:

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A) Example traces of sEPSC recordings from a CeA neuron during baseline conditions without pretreatment (left, n=15 cells from 13 mice) or with pretreatment of fluorocitrate (middle, n=13 cells from 7 mice) or minocycline (right, n=14 cells from 7 mice) and after bath application of 20mM EtOH in all conditions. B) Summary time-course of the effect of 20mM EtOH in vehicle, 100μM fluorocitrate and 100μM minocycline conditions. C) Summary of the peak effect of 20mM EtOH on sEPSC frequency in CeA neurons normalized to baseline (typically between min 14–18). D) No sex differences in the effect of 20mM EtOH on sEPSC frequency in CeA neurons normalized to baseline. E) Summary of the effect of 20mM EtOH on sEPSC amplitude in CeA neurons normalized to baseline. F) Pretreatment with 100mM fluorocitrate reduced uptake of 1mM SR101 in the CeA compared to vehicle condition. G) Summarized data of SR101 staining in vehicle and 100mM fluorocitrate treatment conditions from 3 males and 3 females (data points represent average from 2 slices per mouse under each condition). In all graphs, * indicates significant difference between conditions. # indicates significant difference from baseline. Individual points represent neurons.

Table 1: Pretreatment conditions did not alter baseline sEPSC frequency, amplitude, or cell capacitance.

One-way ANOVA indicated there were no differences in basal amplitude, frequency, and membrane capacitance in cells recorded between the treatments. Two-way ANOVA indicated there were no sex differences between the treatment groups.

Group Number of cells Basal Frequency Basal Amplitude Membrane Capacitance Sex difference?
Vehicle 29 2.32±0.52 −8.65±0.37 66.10±5.82 No
Fluorocitrate 28 1.83±0.39 −9.12±0.66 84.96±8.46 No
Minocycline 28 3.07±0.59 −8.48±0.55 80.03±7.77 No
GFAP DREADDs Vehicle 15 1.81±0.32 −8.32±0.48 81.00±7.94 No
GFAP DREADDSs CNO 13 1.83±0.34 −8.42±0.51 87.00±9.80 No
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Chemogenetic astrocyte inhibition attenuates EtOH effects on glutamatergic transmission

The above data suggests the critical involvement of astrocytes in mediating the effect of EtOH on glutamatergic transmission. Therefore, to explore this mechanism further, we bilaterally microinjected AAV-GFAP-hM4D(Gi)-mCherry into the CeA to be able to chemogenetically target CeA astrocytes (fig. 2A). The mice were then utilized for whole-cell patch clamp electrophysiology to determine if chemogenetic astrocyte modulation via CNO application is able to reduce the ability of 20mM EtOH to enhance sEPSC frequency in the CeA. Slices were pretreated with 10μM CNO in ACSF for 20–30 mins with CNO concentration being maintained during subsequent recordings of EtOH applications. As a control, virally injected slices from the same animal were used, and EtOH application experiments were performed without the presence of CNO in the ACSF. In control slices, 20 mM EtOH significantly increased sEPSC frequency (30.12±7.206% increase from baseline, n=15, t=4.180, p=0.0009, fig. 2C) with no significant effect on sEPSC amplitude (−1.811±2.842% from baseline, n=14, t=0.6373, p=0.5350, fig. 2E). CNO pretreatment significantly (t=2.794, p=0.0096, fig. 2B, C) reduced the ability of 20mM EtOH to increase sEPSC frequency (1.772±7.039% from baseline, n=13, t=0.2518, p=0.8055), with no effect on sEPSC amplitude (1.747±4.430% from baseline, n=13, t=0.3943, p=0.7002, fig. 2E). No sex differences were seen in these effects (F(1,24)=0.03857, p=0.8460, fig. 2D), therefore, the combined data in fig. 2C was utilized for conclusions. These data, together with the findings in figure 1, support the hypothesis that EtOH acts through astrocytes to increase glutamatergic transmission in the CeA.

Figure 2: Chemogenetic inhibition of astrocytes reduces the ability of EtOH to enhance glutamatergic transmission in the CeA.

Figure 2:

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A) Representative confocal images showing injection site confirmation in the CeA and colocalization of mCherry from GFAP-hM4D-Gi-DREADD virus with the astrocytic marker S100. B) Example traces of sEPSC recordings from CeA neurons without pretreatment (Vehicle, left, n=15 cells from 10 mice) or with CNO pretreatment (right, n=13 cells from 9 mice) during baseline and during the 20mM EtOH application peak effect. C) Summary data of 20mM EtOH and CNO application on CeA sEPSC frequency. * indicates significant difference between the groups. # indicates significant difference from baseline. D) Summary data showing no sex differences in 20mM EtOH and CNO application effects on CeA sEPSC frequency. E) Summary data showing no effect of 20mM EtOH and CNO application on sEPSC amplitude. Individual points represent neurons.

Lipopolysaccharide (LPS) modulation of glutamatergic transmission in the lateral CeA

The above experiments suggest that EtOH acts on the CeA glutamatergic transmission through astrocytic signaling. We next sought to examine whether direct neuroimmune activation might also increase sEPSC frequency in the CeA. To that end, we utilized lipopolysaccharide (LPS, 500ng/ml) – the gram-negative bacterial endotoxin commonly used to induce neuroinflammation in rodent models – to determine if any effects of LPS could be blocked by fluorocitrate or minocycline pretreatment. Although there was a trend toward an LPS-induced increase in sEPSC frequency from baseline under vehicle conditions at the concentration tested, this did not reach significance in the one-sample t-test (20.29±9.923% from baseline, n=14, t=2.034, p=0.0617, fig. 3A, B). However, LPS significantly increased sEPSC frequency following minocycline pretreatment (26.85±10.12% from baseline, n=15, t=2.654, p=0.0189), suggesting that microglia activation may limit overall LPS effects on CeA glutamatergic transmission at the concentration tested. Importantly, LPS application in the presence of fluorocitrate did not significantly alter sEPSC frequency from baseline (−8.417±8.391% from baseline, n=14, t=1.003, p=0.3341, fig. 3A, B) suggesting astrocyte activation is necessary for LPS effects on CeA glutamatergic transmission in perhaps a similar mechanism required for the effects of EtOH under these recording conditions. Overall, one-way ANOVA indicated a significant difference between the pretreatments (F(2,40)=3.845, p=0.0297), with Tukey’s post-hoc analysis showing a significant difference between the fluorocitrate and minocycline treated groups (p=0.0319).

Figure 3: LPS modulates glutamatergic transmission in the CeA.

Figure 3:

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A) Example traces of sEPSC recordings from CeA neurons during baseline conditions without pretreatment (Vehicle, left, n=14 cells from 10 mice), with fluorocitrate pretreatment (middle, n=14 cells from 8 mice), or with minocycline pretreatment (right, n=15 cells from 9 mice) and 500ng/mL LPS application. B) Summary data of sEPSC frequency with 500ng/mL LPS application by itself, with fluorocitrate, and with minocycline C) Summary data showing no significant sex differences in sEPSC frequency with 500ng/mL LPS application by itself, with fluorocitrate, or with minocycline. D) Summary data of the effect of 500ng/mL LPS application by itself, with fluorocitrate, or with minocycline on sEPSC amplitude. In all the graphs * indicates a significant difference between indicated groups and # indicates significant difference within a group compared to its baseline. Individual points represent neurons.

Additionally, two-way ANOVA revealed no significant sex differences in the effect of 500ng/mL LPS on sEPSC frequency (F(2,37)=2.246, p=0.1424, fig. 3C); however, in the pooled dataset we noticed a large coefficient of variation in the fluorocitrate treated group (373.0%) compared to vehicle (183%) and minocycline (145.9%) treated groups. Therefore, to explore this variability further, we examined effects of LPS on sEPSC frequency across pretreatment conditions within each sex separately. The results indicated that in males, fluorocitrate pretreatment led to a significant LPS-induced reduction of sEPSC frequency compared to baseline (−26.92±6.538% from baseline, n=7, t=4.118, p=0.0062). Additionally, in males, a one-way ANOVA showed a significant difference between pretreatment conditions (F=5.656, p=0.0131), with fluorocitrate treated cells being significantly different both from LPS with vehicle (p=0.0289) and minocycline pretreatment (p=0.0234). In females, fluorocitrate application was not significantly different from baseline within or between any pretreatment group (F=0.5053, p=0.6105), however, this is likely due to the large variability in the data with cells from females having 355.0% variability in the fluorocitrate treatment condition, compared to 148.5% in vehicle and 170.5% in minocycline treated groups. In comparison, fluorocitrate-treated slices from males had 64.25% variability, with vehicle having 266.2%, and minocycline having 160.4% variability. None of the treatment conditions altered the sEPSC amplitude compared to baseline or each other (F(2,41)=0.4260, p=0.6560, fig. 3D), and no sex differences were found (F(1,38)=0.03018, p=0.8630). Since blocking of astrocytes, but not microglia, abolished the effects of LPS on sEPSC frequency, we interpret these data to suggest that LPS modulation of glutamatergic transmission in the CeA is largely dependent on astrocyte function. Further at the concentration tested, LPS effects may be limited by microglia activation in these studies.

Discussion

Previous research indicates that acute EtOH can increase glutamatergic transmission in the lateral subdivision of the CeA (Silberman et al 2015) but the mechanism by which this occurs has yet to be fully elucidated. One potential mechanism is via EtOH modulation of neuroimmune signaling (Bajo et al., 2015, 2014; Nwachukwu et al., 2021; Patel et al., 2019; Varodayan et al., 2018). To that end, this study aimed to examine the role that microglia and astrocytes may play in the effects of EtOH on CeA glutamatergic transmission. The results suggest that EtOH increases glutamatergic transmission in the CeA as shown by an increase in the sEPSC frequency (fig. 1). The ability of EtOH to enhance CeA glutamatergic transmission was significantly ameliorated by pretreating with fluorocitrate – an astrocyte inhibitor – but not minocycline – a microglia inhibitor (fig. 1). Chemogenetic inhibition of astrocytes in the CeA further supported the hypothesis that astrocytes play a role in EtOH effects on CeA glutamatergic transmission (fig. 2). To our knowledge, this is the first study to directly examine the interaction of microglia and astrocytes with EtOH in the CeA glutamatergic transmission utilizing brain slice electrophysiology with pharmacologic and chemogenetic approaches. We further sought to examine whether direct neuroimmune cell activation with LPS would result in similar changes to CeA glutamatergic transmission. The results showed that LPS modulation of glutamatergic transmission in the CeA also seems to be mediated by astrocytes. The data also suggest that microglia activation may limit LPS effects on CeA glutamatergic transmission as pretreatment with minocycline uncovered a significant stimulatory effect of the 500ng/ml concentration of LPS in these studies (fig. 3).

Glutamatergic neurotransmission in the CeA has been implicated in the reinforcing actions of EtOH (Varodayan et al., 2017). EtOH induced dysregulation of the glutamate system in the CeA has been shown to contribute to hyperexcitability and craving in EtOH withdrawal (Roberto et al., 2020). Generally, acute EtOH is thought to inhibit glutamate transmission in most brain regions (Roberto et al., 2020); however, the current findings indicate that acute EtOH increased glutamatergic transmission in the lateral subdivision of the CeA, supporting previous studies (Silberman et al., 2015). This discrepancy between findings from our lab and other findings can be due to multiple factors, including the type of animal used, the subregion of the CeA being studied, and the type of recordings. For example, it has been suggested that in EtOH-preferring P rats, EtOH acts on the postsynaptic glutamatergic receptors, such as mGluRs and NMDA receptors, increasing their expression (Obara et al., 2009; Roberto et al., 2020). Our studies used C57Bl/6J mice and saw changes only in glutamatergic transmission frequency and not amplitude (fig. 1), suggesting a presynaptic effect. However, the current study examined sEPSC, while other studies have shown that acute EtOH inhibits miniature EPSCs, suggesting EtOH enhancement of glutamatergic transmission may be reliant on activity-dependent neurotransmitter release. Additionally, our study focused particularly on the lateral subdivision of the CeA, which has different synaptic connections than the medial CeA with a higher density of neuropeptides, input from the cortex and the thalamus, and projections to the bed nucleus of stria terminalis and substantia inominata (Roberto et al., 2012). Other work has shown that acute EtOH increases GABAergic neurotransmission in the medial subdivision of the CeA (Bajo et al., 2008; Cruz et al., 2011; Roberto, 2004). Since neurons in the lateral CeA provide GABAergic projections to the medial CeA, it is possible that the enhanced glutamatergic transmission seen in these studies is a direct mechanism related to enhanced GABAergic transmission in the medial CeA. Future studies will be necessary to further examine these discrepancies and potential intra-CeA microcircuit mechanisms.

In the current study, we found that EtOH enhancement of CeA glutamatergic transmission required astrocyte signaling but did not require microglia signaling. Both pharmacologically and chemogenetically inhibiting astrocyte function blocked the ability of acute EtOH to increase CeA glutamatergic transmission. The vast majority of neurons in the CeA are GABAergic medium spiny neurons, suggesting that the likely source of glutamatergic neurotransmission in the CeA may be from glutamatergic afferents from the basolateral amygdala and other brain regions implicated in alcohol use disorders (Krettek and Price, 1978; Pitkänen et al., 1995; Savander et al., 1995). The results of the current study indicate that neuroimmune cells, particularly astrocytes, may be critical regulators or local sources of glutamatergic transmission in the CeA. Previous research has also shown that astrocytes are key regulators of glutamatergic transmission in many brains regions (Murphy- Royal et al., 2017), and it is possible that the form of glutamatergic transmission studied here, amenable to EtOH modulation, may be a direct response of astrocytic glutamate release. It remains to be seen how chronic EtOH might alter this astrocyte-mediated mechanism, but both acute and chronic EtOH exposure have been shown to modulate the activity of astrocytic excitatory amino acid transporters in other brain regions (Ayers-Ringler et al., 2016; Griffin III et al., 2014; Mulholland et al., 2009), suggesting the mechanism of action seen here may be critical to EtOH effects throughout the central nervous system. Additionally, neuroinflammation appears to be a key contributor to EtOH actions in many brain regions, and previous research indicates neuroimmune signaling plays a key role in EtOH modulation of CeA GABAergic transmission (Bajo et al., 2014). Previous research indicates that the expression of cytokines, such as TNFα, IL-1β, NF-κB, and MCP-1, is enhanced after a single EtOH exposure, suggesting involvement of neuroinflammation in the effect of EtOH in the brain (Qin et al., 2008; Robinson et al., 2014). Studies have shown that increased neuroinflammation through LPS injections can lead to increased EtOH consumption, suggesting that neuroinflammatory pathways are involved in the behavioral effects of EtOH (Blednov et al., 2011), possibly by increased expression of proinflammatory cytokines (Robinson et al., 2014). It is worth noting that the effects of astrocytes on glutamatergic transmission in the CeA after acute EtOH exposure may be due to non-neuroinflammation pathways, such as increased calcium signaling, modified gap junction permeability, modulation of glutamate transporters, and glutamate-glutamine cycle (Adermark and Bowers, 2016; Tani et al., 2014).

It should also be noted that many of the acute effects of EtOH on CeA GABA and glutamatergic transmission are also mediated via corticotropin releasing factor (CRF) signaling (Guglielmo et al., 2017; Silberman et al., 2015; Varodayan et al., 2017). Although the precise mechanism by which EtOH-CRF- and EtOH-astrocyte-signaling in the CeA interact is not fully known, some studies suggest EtOH modulation of CRF levels increases neuroimmune signaling in various brain regions in a feed-forward regulatory mechanism, at least in terms of GABA receptor subunit function (Aurelian and Balan, 2019). Determining the mechanism(s) of interaction between EtOH, CRF, astrocyte, and glutamate transmission in the CeA is likely an important step in terms of development of alcohol use disorder treatments. Future research will be necessary to examine this hypothesis.

Our findings, however, did not show a requirement of microglia signaling in mediating the neuroimmune-related effects of acute EtOH. This result was surprising as microglia have been implicated in EtOH intake behaviors, and microglia mediated EtOH-induced neuroinflammation in response to excessive EtOH consumption is critical for EtOH-induced neurodegeneration (Melbourne et al., 2019). It is possible that chronic EtOH exposure may be needed to induce microglia-dependent EtOH modulation of synaptic transmission in the lateral CeA that would not be seen with acute application studies in previously naïve animals. Indeed, recent studies by Warden et al., 2020 showed that microglia depletion results in a significant reduction of presynaptic glutamatergic transmission in the medial subdivision of the CeA only in EtOH dependent mice with no effect of microglia depletion on sEPSC frequency in non-dependent mice. Interestingly, studies have shown that the acute effect of EtOH on neuroimmune gene expression varies depending on the method, the timing, and brain region, with some studies showing an increase in cytokine expression and others showing a decrease or no change (Melbourne et al., 2019; Qin et al., 2008). In the current study, we used minocycline to inhibit microglia, which has its limitations. Minocycline has been shown to attenuate the induction of expression of M1 pro-inflammatory microglia markers but does not affect the expression of M2 anti-inflammatory microglia (Kobayashi et al., 2013). Interestingly, minocycline administration in C57Bl/6J mice caused a significant reduction in EtOH drinking (Agrawal et al., 2011); however, a clinical study in heavy drinkers did not see an effect of minocycline in EtOH-induced craving or serum cytokine levels (Petrakis et al., 2019). Indeed, the interaction between EtOH and microglia dependent signaling is complex (Melbourne et al., 2019). Studies have shown that even after microglia depletion, there is a significant upregulation in astrocytic gene expression after EtOH exposure, suggesting that microglia and astrocytes may be important effector cells for regulating the actions of EtOH, although this was not directly examined in the CeA (Warden et al., 2021). Therefore, the effects of microglia and astrocytes on EtOH induced increase in glutamatergic transmission in the CeA in models of dependent vs non-dependent EtOH exposure and intake models needs to be further examined.

In summary, the current study showed that acute EtOH enhances lateral CeA glutamatergic transmission through an astrocyte mediated mechanism. The precise mechanism of action of astrocytes on the acute EtOH mediated increase of glutamatergic transmission in the CeA is unknown and needs to be investigated in future studies. The acute effects of EtOH on this form of glutamatergic transmission did not appear to be altered by pharmacologic inhibition of microglia. The potential for microglia-astrocyte interactions on EtOH modulation of CeA glutamatergic transmission following chronic EtOH administration, and whether targeting CeA neuroimmune cells as a treatment for alcohol use disorders remains a critical open question to be addressed in future studies.

  • Ethanol increases glutamatergic transmission in the lateral central amygdala

  • Pharmacologic and chemogenetic astrocytic inhibition reduced ethanol effect

  • Astrocytes are required for ethanol modulation of glutamatergic transmission

  • Microglia are not required for ethanol modulation of glutamatergic transmission

Acknowledgements

The authors would like to acknowledge Bailey Keller, Victoria Vernail, and Kaitlin Carson for their support with immunohistochemistry and discussions of the data. This work was funded by NIH grants AA026865 and AA027943, and PA Options for Wellness Student Award.

Footnotes

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