Chemogenetic manipulation of astrocytic signaling in the basolateral amygdala reduces binge-like alcohol consumption in male mice

Abstract

Binge drinking is a common occurrence in the United States, but a high concentration of alcohol in the blood has been shown to have reinforcing and reciprocal effects on the neuroimmune system in both dependent and non-dependent scenarios. The first part of this study examined alcohol’s effects on the astrocytic response in the central amygdala and basolateral amygdala (BLA) in a non-dependent model. C57BL/6J mice were given access to either ethanol, water, or sucrose during a “drinking in the dark” paradigm, and astrocyte number and astrogliosis were measured using immunohistochemistry. Results indicate that non-dependent consumption increased glial fibrillary acidic protein (GFAP) density but not the number of GFAP+ cells, suggesting that non-dependent ethanol is sufficient to elicit astrocyte activation. The second part of this study examined how astrocytes impacted behaviors and the neurochemistry related to alcohol using the chemogenetic tool, DREADDs (designer receptors exclusively activated by designer drugs). Transgenic GFAP-hM3Dq mice were administered clozapine N-oxide both peripherally, affecting the entire central nervous system (CNS), or directly into the BLA. In both instances, GFAP-Gq-signaling activation significantly reduced ethanol consumption and correlating blood ethanol concentrations. However, GFAP-Gq-DREADD activation throughout the CNS had more broad effects resulting in decreased locomotor activity and sucrose consumption. More targeted GFAP-Gq-signaling activation in the BLA only impacted ethanol consumption. Finally, a glutamate assay revealed that after GFAP-Gq-signaling activation glutamate concentrations in the amygdala were partially normalized to control levels. Altogether, these studies support the theory that astrocytes represent a viable target for alcohol use disorder therapies.

1 |. INTRODUCTION

Although alcohol consumption is socially accepted and binge drinking is sometimes considered an American pastime, the problems associated with excessive binge drinking create a substantial burden for the United States and internationally (Bloomfield et al., 2003; Grittner et al., 2020). These problems include, but are not limited to, public safety concerns, interpersonal relationship issues, and saddling the resources of our health care system (Schomerus et al., 2011). With one in six adults participating in binge drinking episodes multiple times a month (CDC, 2013; Esser et al., 2014), the negative consequences that arise from excessive alcohol consumption are estimated to cost the United States approximately $250 billion a year (Sacks et al., 2015). Such repetitive bouts of high blood alcohol levels fundamentally change neurobiological functions and can lead to neuroplastic events that contribute to the development of alcohol use disorders (AUDs) (Der-Avakian & Markou, 2012; George et al., 2012; Markou et al., 1998). These maladaptations represent potential therapeutic targets that may curb excessive alcohol consumption and reduce the impact of alcohol misuse on society. Although there are a variety of alcohol-induced neuroplastic changes, a growing body of evidence suggests that the neuroimmune system undergoes such alcohol-induced neuroplasticity and that dysregulation of the neuroimmune system is influential in addictive behaviors (Coller & Hutchinson, 2012; Crews, 2012; Crews et al., 2011, 2015).

Much of the work in the AUD field has focused on how the neuroinflammatory response elicited by alcohol may mediate brain damage in dependent models (Crews & Vetreno, 2014). However, the neurobiological consequences of ethanol misuse in non-dependent episodic binge drinking can vary significantly from those observed in dependence (Koob & Schulkin, 2019), including the neuroimmune system (Grifasi et al., 2019). Moreover, neuroimmune responses have the propensity to have behavioral consequences independent of brain damage (Coller & Hutchinson, 2012; Hutchinson & Watkins, 2014). In fact, we have recently shown that binge-like consumption drives a pro-inflammatory cytokine response in the amygdala that can be manipulated to reduce consumption (Marshall, Casachahua, et al., 2016; Marshall et al., 2017), but the influence of glial cells on binge-like consumption is still elusive. The development of chemogenetic tools that specifically target GFAP+ cells has allowed for a more discrete determination of the role of astrocytes in a variety of behaviors (Agulhon et al., 2013; Roth, 2016), including reward (Bull et al., 2014; Scofield et al., 2015). Astrocytes are a key component of the neuroimmune system and its response but can also act as mediators to behavioral correlates. Perturbation of astrocytes has the potential to regulate various aspects of alcohol’s effects on the central nervous system (CNS) including blood-brain barrier disruption, increased proinflammatory cytokine production, and dysregulation of glutamatergic tone (Ayers-Ringler et al., 2016; Lacagnina et al., 2016; Miguel-Hidalgo, 2018). Both clinical and preclinical studies indicate that a history of alcohol abuse can lead to astrocyte dysregulation, but the astrocytic response varies depending on the brain region assessed, the concentration of ethanol, and the duration of ethanol exposure (Bull et al., 2015). Previous work has shown the influence of the amygdala on ethanol consumption (Gilpin et al., 2015; Roberto et al., 2012), and the role of neuroimmune dysregulation in the region (Bajo, Herman, et al., 2015; Bajo, Varodayan, et al., 2015; June et al., 2015; Marshall, Casachahua, et al., 2016; Marshall et al., 2017). This study is an extension of that work and will determine the effects of non-dependent ethanol exposure on the astrocytes of the amygdala as well as how those amygdalar astrocytes may influence consummatory behaviors.

To study non-dependent consumption and the reciprocal relationship with astrocytes, this study uses the drinking in the dark (DID) model. This model is unique because rodents readily and voluntarily consume binge levels of ethanol leading to neurobiological changes that can be manipulated to study their influence on consumption (Rhodes et al., 2005; Thiele & Navarro, 2014). Importantly, this model does not induce brain damage or dependence representing an important group of individuals with AUDs who are neither dependent nor show evidence of brain damage (Bobak et al., 2004; Cox et al., 2013; Epstein et al., 2004; Marshall, Casachahua, et al., 2016; Sprow et al., 2015). To understand astrocytic signaling’s role in alcohol consumption, this research utilizes designer receptors that are exclusively activated by designer drugs (DREADDs) encoded under a GFAP promoter. The DREADD virus incorporates an artificial muscarinic receptor that has no natural endogenous ligands and is only activated by targeted exogenously administered compounds (Lee et al., 2014; Roth, 2016). Incorporating the virus with a GFAP promoter, affords the opportunity to separate the influence of astrocytes from other neuroimmune modulating cells including microglia, neurons, and endothelial cells in the microenvironment of the amygdala (Agulhon et al., 2013; Bull et al., 2014; Lee et al., 2014). This work tested the hypothesis that alcohol alters the astrocyte population of the amygdala and that those astrocytes can be manipulated to impact ethanol consumption.

2 |. METHODS

2.1 |. Animals

All animal manipulations were approved by both the North Carolina Central University and High Point University Institutional Animal Care and Use Committees following the procedures established in the Guidelines for the Care and Use of Laboratory Animals (NRC, 2011). In this study, male GFAP-hM3Dq transgenic (MGI:6148045), littermates, and C57BL/6J (IMSR_JAX:000664) mice were used. A colony of GFAP-hM3Dq mice was established from initial breeders provided courtesy of the laboratory of Dr. Ken McCarthy (University of North Carolina-CH, Chapel Hill, NC) (Agulhon et al., 2013; Xie et al., 2015, 2017). Male C57BL/6J breeders from (Jackson Laboratories; Bar Harbor, ME) are periodically introduced to maintain genetic variability. All progeny were tested for the presence of the genes for hM3Dq and the Tg tet R fluorescent tag. All animals were group housed in a vivarium maintained at 22°C with a reversed 12:12 hr light:dark cycle prior to any experimental manipulations. A week before behavioral studies and/or immediately after surgical manipulations, mice were transitioned to individual housing. Mice had ad libitum access to Teklad Diet® 7912X (Harlan Laboratories Inc.; Indianapolis, IN) and water, unless otherwise stated (Marshall et al., 2015).

2.2 |. Drinking in the dark paradigm

Binge-like drinking was modeled using the “drinking-in-the-dark” paradigm (Rhodes et al., 2005; Thiele & Navarro, 2014). Approximately 3 hr into the dark–light cycle, home cage water bottles were switched out for bottles containing 20% (v/v) ethanol, 3% (w/v) sucrose, or tap water. Access to the varying solutions was given for a time period of 2 hr for four consecutive days. For mice that underwent multiple DID cycles, 3 days separated each cycle to model periodic binge-like consumption (Marshall et al., 2015).

Tail blood samples (~50 μl) were obtained from the lateral tail vein to determine blood ethanol concentration (BEC). BECs (mg/dl) were quantified from serum samples run in duplicate using the colorimetric assay, EnzyChrom™ Ethanol Assay Kit (BioAssay Systems; Hayward, CA) (Grifasi, Evans, et al., 2019; Jeong et al., 2005).

2.3 |. Immunohistochemistry

Immunohistochemistry (IHC) with anti-GFAP rabbit polyclonal antibody (Abcam; ab7260; RRID:AB_305808) was utilized to characterize the astrocytic response following alcohol exposure in C57BL/6J mice as well as to determine the fidelity of DREADD expression in astrocytes of transgenic mice. Fluorescent immunohistochemical procedures were used to colabel DREADD and GFAP, but anti-GFAP was conjugated to 3,3′-Diaminobenzidine (DAB) for astrogliosis measures. Immunohistochemical procedures were similar to our previous publications (Grifasi, Evans, et al., 2019; Grifasi, McIntosh, et al., 2019; Marshall et al., 2016). For astrogliosis studies, mice were split into groups undergoing one cycle (water n = 5, sucrose n = 7; ethanol n = 7) or three cycles (water n = 5; sucrose n = 6; ethanol n = 8) of DID. Following euthanasia via transcardial perfusion of paraformaldehyde, brains were extracted and sliced at 40μm using a Compresstome VF-300 (Precisionary Instruments; Greenville, NC) (Abdelaal et al., 2015). Sections were stored in cryopreserve (1% (w/v) polyvinyl-pyrrolidone; 50% (v/v) 0.2 M PBS and ethylene glycol) in a 1:4 series. Brain sections were washed prior to a 30-min blocking incubation to reduce nonspecific antibody binding in a 3% goat serum/0.2% Triton X/PBS solution. For DAB IHC, additional steps with 1% H₂O₂ and washes were used to quench endogenous peroxidase prior to the blocking step (Marshall et al., 2013). After blocking, sections were incubated in a Rb anti-GFAP primary antibody (Abcam, 1:500) for 48 hr at 4°C. The antibody has previously been characterized with appropriate controls by the manufacturer. The primary antibody was then removed with a series of PBS washes. For the astrocytic activation study, the primary antibody was tagged with a goat anti-rabbit biotinylated secondary (Vector Labs) before the horseradish peroxidase enzymatic conjugation with DAB (Millipore Sigma, St. Louis, MO). Tissue sections from the water, sucrose, and ethanol groups were all run simultaneously for DAB analysis. Processed sections were mounted onto glass slides and coverslipped with CYTOSEAL™ 60 (RICHARD-ALLAN SCIENTIFIC; Kalamazoo, MI).

For immunofluorescence, a subset of transgenic animals (n = 4) were utilized to determine the fidelity of the DREADD construct in GFAP+ cells. Following serial washes in PBS, nonspecific binding was blocked using 3% goat serum before the tissue was incubated in chicken anti-GFAP (1:1,000; Millipore; RRID:AB_177521) and rabbit anti-mCherry (1:250; Millipore; RRID:AB_2889995) for 48 hr at 4°C. Following a series of washes in PBS, fluorescent secondary antibodies (Invitrogen, Carlsbad, CA, USA; 1:1,000) were added and the tissue was incubated for 2 hr in goat anti-chicken Alexa Fluor ® 488 (RRID:AB_2534096) and goat anti-rabbit Alexa Fluor ® 568 (RRID:AB_143157). After the secondary was removed, tissue was mounted and sealed using Vectashield® Plus Anti-fade mounting medium with DAPI (Vector; RRID:AB_2336790). All antibodies used have been summarized in Table 1.

TABLE 1.

Descriptive table of primary antibodies used in experiments

Antibody	Immunogen	Host	Source	RRID	Concentration
Anti-GFAP	Purified glial filament protein	Rabbit	Abcam	AB_305808	1:500
Anti-GFAP	Purified glial filament protein	Chicken	Millipore	AB_177521	1:1,000
Anti-mCherry	His-tagged full length recombinant mCherry from Discosoma sp (mushroom anemone)	Rabbit	Millipore	AB_2889995	1:250

2.4 |. Immunohistochemistry quantification

Images of the amygdala were captured using a 10× objective on an Olympus BX-51 microscope (Olympus, Center Valley, PA, USA) linked to a Q Color 3 (Olympus; Q-Capture ProTM software) for DAB-labeled images. For DAB photomicrographs, subregions of the amygdala (BLA and CeA; between Bregma −0.58 mm and −2.36 mm) were individually traced on each image. Astrocytes were quantified using densitometric analysis as well as profile cell counts. Experimenters were blind to the treatment groups because all animal identifications were coded during imaging. GFAP+ pixel densitometry was assessed using experimentally determined threshold densitometry in CellSens (Olympic; Tokyo, Japan). Immunopositive pixels within each focal region was determined by automated counts of the pixels within a region of interest (Marshall, Casachahua, et al., 2016; Marshall et al., 2013). The DAB immunoreactive density was quantified in pixels per defined area (pixels/mm²). GFAP+ cells were counted within each region and is expressed as GFAP+ cells/section. Four animals were removed because less than seven usable sections were analyzed.

To examine the fidelity of the DREADD construct with GFAP+ cells, photomicrographs of the BLA and hippocampus were captured using a Zeiss LSM 800 Laser Confocal Microscope (Zeiss, PA, USA) with a linked digital camera, Nikon Eclipse Ti inverted microscope and Nikon Eclipse C1. Z-serial images at 1.5 μm were collected and analyzed using the LSM Image Examiner software.

2.5 |. Surgeries

Animals used to understand the role of astrocytic GPCR signaling in the basolateral amygdala were implanted with bilateral guide cannulae (Plastics One; Roanoke, VA), similar to our previous studies (Marshall, Casachahua, et al., 2016; Marshall et al., 2017). Briefly, following i.p. injections (1.5 ml/kg) of an anesthetizing cocktail of xylazine (10mg/kg) and ketamine (100mg/kg), cannulae were lowered into the BLA (AP: −1.22, ML: ±3.01, DV: −4.75) (Paxinos & Franklin, 2004). Following surgery, mice were given at least a week before behavioral testing. At the conclusion of behavioral studies, placements were histologically verified using Alcian blue dye (0.3 μl/injection site).

2.6 |. Consumption study

The effect of GFAP-DREADD activation was measured during the DID to determine the role of astrocytic GPCR signaling in ethanol consumption. GFAP-DREADDs were activated throughout the CNS and directly in the amygdala. Group sizes were based on transgenic and littermate availability. To highlight the role of astrocytes in the CNS, GFAP-DREADD transgenic mice (n = 9), littermate controls (n = 8), or WT (C57BL/6J; n = 4) were given systemic clozapine-N-oxide (CNO; 3mg/kg, IP) for two cycles of DID but received vehicle (1% dimethyl sulfoxide in 0.9% saline) during the third test day. CNO or vehicle was administered 20–40 min prior to the DID similar to our previous studies (Rinker et al., 2017). Finally, GFAP-Gq-DREADD activation effects on sucrose were tested to determine the selectivity of astrocyte effects.

Only GFAP-DREADD transgenic mice (n = 19) were used to determine if site-directed manipulations in the BLA altered ethanol consumption. We used a Latin-square 2 × 2 design, so that animals received both CNO and vehicle serving as their own control. The study started with 24 animals, however, five were excluded for missed location and/or cannula loss between trials. CNO (900 pmol;0.3 μl) or vehicle was bilaterally administered into the surgically placed guide cannulae, and infusions were administered using a Hamilton syringe (Reno, NV) and Harvard Apparatus PHD 2000 infusion pump (Holliston, MS) at a rate of 0.15 μl/min. After infusion, injectors remained in guide cannulae for an additional 3 min to allow for proper diffusion. Consumption was normalized to body weight and expressed as g of ethanol/kg of body weight. Sucrose consumption was then tested in the same animals, but the test was only run for one DID cycle.

2.7 |. Open field tests

Alcohol naïve GFAP-DREADD transgenic mice, WT, and their littermates were tested in an open field task (Marshall, Casachahua, et al., 2016; Marshall et al., 2017). Open field tests (OFTs) were performed with minimal illumination in the room 3 hr into the dark cycle, similar to their home cage vivarium. A 2 × 2 Latin-square Design was used to test the effects of GFAP-DREADD activation throughout the CNS (GFAP-DREADD n = 7, Littermate = 6, WT = 5) and in the BLA (GFAP-DREADD n = 7, WT = 7) on locomotor and anxiety-like behaviors. Similar to our consumption, CNO or vehicle was administered 20–40 min prior to access to the OFT. The movement and position of mice in open field arenas from Med Associates (27.31 × 27.31 × 20.32 cm; St Albans, VT) were tracked with Activity Monitor 7 software (Med Associates). Each session was measured over a 2-hr period. Time spent in the center of the open arena was used as a correlate of anxiety-like behavior (Prut & Belzung, 2003). Locomotor data are expressed as time active (seconds) as well as distance traveled (cm), but anxiety was assessed using time spent in the center of the arena (seconds).

2.8 |. Glutamatergic tone assay

The day after three cycles of DID ethanol exposure mice were divided up into two groups. In group 1, C57BL/6J (n = 6) or GFAP-Gq-DREADD (n = 5) mice were given alcohol by intragastric gavage (1.5g/kg; 20% w/v ethanol) to reach BECs similar to the binge paradigm (Chen et al., 2013; Marshall et al., 2015; Walker & Ehlers, 2009) (Chen et al., 2013). In the control group 2, C57BL/6J (n = 5) and GFAP-Gq-DREADD (n = 5) mice received water. However, all mice were given CNO i.p (3mg/kg) prior to gavage and euthanized by rapid decapitation. The amygdala was microdissected out from fresh brains and processed according to the manufacturer’s protocol in the Glutamate Assay Kit (abcam; ab83389). Each sample was run in duplicate and the concentration was determined by comparison to standards using a Spectramax M2 microplate reader. Glutamate concentrations were normalized to protein levels determined by BCA protein assay and are expressed as nM Glutamate/g of protein (Marshall et al., 2013).

2.9 |. Statistical analyses

All data were graphed and analyzed in GraphPad Prism 8 (GraphPad Software, Inc. La Jolla, CA). Two-way analyses of variance (ANOVAs) were used to analyze GFAP immunoreactivity, cell counts, ethanol consumption, blood ethanol concentrations, daily sucrose consumption, glutamate concentrations, and locomotor activity in the open field test. Post hoc Bonferroni tests were only run if there was an interaction or if a main effect of CNO treatment or ethanol exposure was observed. All data are reported as the mean ± standard error of the mean and considered significant if p < 0.05, two-tailed.

3 |. RESULTS

3.1 |. Binge-like ethanol consumption increases BLA GFAP immunoreactivity

The parameters of the DID including consumption and BECs are shown in Table 2. Importantly, the number of cycles did not have any effects on consumption or BECs. Astrocytes were assessed using GFAP+ pixel densitometry as a correlate of astrogliosis. Photomicrographs of the BLA suggested that the water (Figure 1a) and sucrose (Figure 1b) groups had lower GFAP immunoreactivity compared with the ethanol (Figure 1c,d) groups. A two-way ANOVA (treatment × number of cycles) revealed no interaction [F_(2,32) = 0.01, p = 0.98] or main effect of duration of cycles [F_(1,32) = 0.13, p = 0.72], but there was a main effect of treatment [F_(2,32) = 12.59, p < 0.001] in the BLA. Post hoc Bonferroni tests revealed that after both one and three cycles of DID ethanol exposure there was a significant increase in GFAP immunoreactivity (Figure 1e). However, in the CeA, there was no interaction [F_(2,32) = 0.13, p = 0.88] or main effects of either treatment [F_(2,32) = 1.32, p = 0.28] or duration [F_(1,32) = 0.62, p = 0.44] according to a two-way ANOVA (Figure 1f). There was not a significant linear correlation between average daily consumption and GFAP immunoreactivity (F_(1,13) = 0.43, p = 0.52, R² = 0.03), but there was a significant positive association between GFAP immunoreactivity and BECs according to a linear regression (F_(1,13) = 5.82, p = 0.03, R² = 0.31; Figure S1).

TABLE 2.

DID parameters for IHC studies

Treatment group	Consumption (g/Kg)	BEC (mg/dl)
Water
1 Cycle	31.3 ± 3.11	-
3 Cycle	39.5 ± 4.21	-
Sucrose
1 Cycle	4.4 ± 0.38	-
3 Cycle	4.6 ± 0.52	-
Ethanol
1 Cycle	4.5 ± 0.33	99.6 ± 9.19
3 Cycle	4.4 ± 0.38	106.2 ± 15.11

Notes: Following DID, consumption (g/kg) and BECs (mg/dl) were analyzed using an unpaired two-tailed t test. T test analysis indicated that there was no main effect of the number of cycles on the water consumption [t(8) = 1.56, p = 0.15], sucrose consumption [t(11) = 0.31, p = 0.77], ethanol consumption [t(13) = 0.34, p = 0.74], or BECs [t(13) = 0.36, p = 0.73].

FIGURE 1.

Glial fibrillary acidic protein (GFAP) is constitutively expressed in both the BLA and CeA as shown in a representative photomicrograph using a 4× objective (a). However, compared with three cycles of sucrose (b), representative photomicrographs of one- (c) and three-cycle ethanol (d) suggest that GFAP is upregulated in the basolateral amygdala of ethanol-exposed mice. White dash lines on the photomicrographs delineate the borders of the BLA. Analysis of GFAP immunoreactivity indicated that the ethanol group had significantly more GFAP positive pixels than the other groups in the BLA (e), but no differences were observed in CeA immunoreactivity (f). Ethanol exposure did not affect the number of GFAP+ cells in either the BLA (g) or CeA (h). Images were taken at 100× magnification. Scale bar in panel a = 1,000 μm; panel d = 200 μm. *p < 0.05 compared to water and sucrose group

Increases in GFAP immunoreactivity can be indicative of morphological changes in astrocytes or changes in the number of astrocytes, so cell counts were conducted to determine if changes in immunoreactivity (IR) were related to astrocyte proliferation. In the BLA, a two-way ANOVA (treatment × cycle number) showed no interaction [F_(2,32) = 0.11, p = 0.90] or main effects of either of treatment [F_(2,32) = 0.67, p = 0.51] or effect of duration of cycles [F_(1,32) = 0.02, p = 0.88] (Figure 1g). According to a two-way ANOVA, the lack of effect of ethanol consumption on astrocyte number was also seen in the CeA (Figure 1h). There was no interaction [F_(2,31) = 1.00, p = 0.37], nor any main effects of either treatment [F_(2,31) = 0.35, p = 0.70] or duration of cycles [F_(1,31) = 0.04, p = 0.84].

3.2 |. Systemic GFAP-Gq signaling reduces ethanol and sucrose consumption

Ethanol consumption was assessed after GFAP-Gq-DREADD activation in the DID paradigm. A two-way repeated measures (RM) ANOVA (genotype × time) revealed no interaction [F_(2,18) = 0.01, p = 0.99] or main effect of time [F_(1,18) = 0.05, p = 0.83], but a significant main effect of genotype [F_(2,18) = 18.63, p < 0.0001] (Figure 2a). Post hoc Bonferroni tests indicated that only the GFAP-DREADD transgenic mice had significantly reduced ethanol consumption compared with both their littermate controls and the C57BL/6J mice. The findings for the BECs were similar to the ethanol consumption, as there was no interaction [F_(2,18) = 0.45, p = 0.64] or main effect of time [F_(1,18) = 0.12, p = 0.73]; however, there was a main effect of genotype [F_(2,18) = 12.40, p < 0.001] according to a two-way RM ANOVA (Figure 2b). Post hoc Bonferroni tests again revealed that only GFAP-DREADD transgenic mice had lower BECs than all other groups. Importantly, when animals were given vehicle in the third DID ethanol cycle, a one-way ANOVA indicated no effect of genotype on ethanol consumption [F_(2,18) = 0.49, p = 0.62] or BECs ([F_(2,18) = 0.008, p = 0.99]; Figure S2).

FIGURE 2.

GFAP-DREADD transgenic animals consumed significantly less ethanol (a) and had lower BECs (b) compared to the littermate control or wild type animals. Gq-DREADD activation also significantly reduced sucrose consumption (c). All data are presented as mean ± SEM. *p < 0.05 compared to the WT and littermate control groups

To test whether the effects of GFAP-Gq-DREADD activation were specific to ethanol, sucrose consumption was also tested over a DID cycle. A two-way ANOVA suggested that there was an interaction between genotype and time [F_(6,54) = 5.37, p < 0.001] as well as a main effect of time [F_(3,54) = 4.52, p = 0.007] but not genotype [F_(2,18) = 2.56, p = 0.10] (Figure 2c). Post hoc Bonferroni tests revealed that only on the fourth day (when CNO was administered) did the GFAP-DREADD mice consume less sucrose than the WT and littermate controls.

3.3 |. Amygdalar GFAP-Gq signaling reduces ethanol but not sucrose consumption

GFAP-DREADD transgenic animals were administered CNO in the BLA and ethanol consumption was assessed. A two-way RM ANOVA (time × treatment) revealed no interaction [F_(1,17) = 0.005, p = 0.95] or main effect of time [F_(1,17) = 0.05, p = 0.83], but there was a significant main effect of CNO treatment [F_(1,17) = 28.45, p = 0.0001] (Figure 3a). Post hoc Bonferroni tests indicated that CNO activation of GFAP-Gq signaling significantly reduced ethanol consumption. This effect was mirrored in the BECs (Figure 3b). There was no interaction [F_(1,17) = 0.21, p = 0.65] or main effect of time [F_(1,17) = 0.09, p = 0.77], however, there was a main effect of CNO treatment [F_(1,17) = 23.31, p < 0.001] according to a two-way RM ANOVA. Importantly, post hoc Bonferroni tests showed that the reduction in BECs was significant, and the average was below the definition of binge drinking. The influence of BLA GFAP-Gq signaling did not extend to sucrose consumption. A two-way RM ANOVA (treatment × time) indicated that there was only a main effect of time [F_(3,51) = 5.14, p = 0.004] but not treatment [F_(1,17) = 0.002, p = 0.97] or an interaction between the variables [F_(3,51) = 0.33, p = 0.80] (Figure 3c).

FIGURE 3.

In the GFAP-DREADD transgenic animals, BLA local injection of clozapine-N-oxide (CNO) induced a decrease in ethanol consumption (a) and BECs in comparison to the vehicle. (b) However, GFAP-Gq-DREADD activation in the BLA did not affect sucrose consumption (c). Panel d shows the approximate location of cannula for the mice used in this study. All data are presented as mean ± SEM. *p < 0.05 compared to vehicle

3.4 |. Systemic but not amygdalar GFAP-Gq signaling impairs locomotor activity

Peripheral administration of CNO to transgenic animals significantly reduced locomotor activity. A two-way RM ANOVA (genetic background × treatment) indicated an interaction [F_(2,15) = 6.68, p < 0.01] as well as main effects of genetic background [F_(2,15) = 7.84, p < 0.001] and CNO administration [F_(1,15) = 5.92, p = 0.03] on the period of active movement (Table S1). Post hoc Bonferroni tests indicate that only when transgenic animals were administered CNO was there a significant reduction in locomotor activity compared with all other genetic groups. A separate two-way RM ANOVA only examining the locomotor activity of animals given CNO (genetic background × time) indicated that there was an interaction [F_(46,345) = 6.65, p < 0.0001] and main effects of time [F_(23,345) = 35.37, p < 0.0001] and genetic background [F_(2,15) = 4.69, p = 0.03] on distance traveled (Figure 4a). Post hoc Bonferroni analysis indicates that the effect of CNO significantly reduced distance traveled during the first 40 min of activity.

FIGURE 4.

Peripheral administration of clozapine-N-oxide (CNO) activated Gq-DREADD signaling throughout the nervous system and significantly decreased locomotor activity in the first 40 min of the open field test (a) as well as the time spent in the arena center in comparisons to the WT (c). However, when CNO was delivered locally and only activated the astrocytes of the BLA there was no impact on locomotion (b) or anxiety-like behavior (d). All data are presented as mean ± SEM. *p < 0.05 compared to the control groups

However, when CNO was administered into the BLA, the two-way RM ANOVA (genetic background × treatment) indicated that there was neither an interaction [F_(1,12) = 0.02, p = 0.88] nor main effects (genotype [F_(1,12) = 0.05, p = 0.83] or CNO treatment [F_(1,12) = 0.11, p = 0.74] observed on locomotion in the OFT (Figure 4b,d) or on time active (Table S1).

3.5 |. GFAP Gq-DREADD activation restores glutamate concentrations in amygdala

Two-way ANOVAs (genotype × treatment) indicated that there was a main effect of both treatment [F_(1,17) = 14.97, p = 0.001] and genotype [F_(1,17) = 15.48, p = 0.001] on glutamate concentrations, but there was no interaction between the two variables [F_(1,17) = 0.008, p = 0.93]. Post hoc Bonferroni analysis indicates that CNO activation of GFAP-Gq signaling increases glutamatergic tone and that ethanol reduces glutamate (Figure 5). However, when GFAP-Gq signaling is activated it partially normalizes the ethanol deficit and is not significantly different than the WT water group.

FIGURE 5.

GFAP-Gq-signaling activation significantly increased glutamate concentrations in the amygdala in the water treated group, but ethanol significantly reduced glutamate. Importantly, in the ethanol group with GFAP-Gq-signaling activation, there was no difference compared with the water WT group. All data are presented as mean ± SEM. *p < 0.05 compared to the WT water control group

4 |. DISCUSSION

The few studies on astrocytes and non-dependent alcohol consumption that are published focus on the effects that alcohol has on the astrocytes (Grifasi, McIntosh, et al., 2019; Mulligan et al., 2011), but this study also examined the effects of altered astrocyte function on alcohol consumption. The major findings of our study are as follows: (a) non-dependent binge drinking causes astrogliosis in the basolateral amygdala, (b) Gq-signaling activation in astrocytes decreases alcohol consumption, and that (c) the GFAP-Gq-DREADD activation ameliorates alcohol-induced decreased glutamate concentration. Taken together, these studies suggest that there is a reciprocal relationship between astrocyte activity and ethanol consumption, wherein ethanol causes astrogliosis but astrocytic receptor signaling reduces alcohol consumption.

4.1 |. Non-dependent binge drinking causes astrogliosis

Alcohol-induced brain damage models have repeatedly reported increased astrocyte activation (Alfonso-Loeches et al., 2010; Hayes et al., 2013; Kelso et al., 2011; Obernier et al., 2002; Qin & Crews, 2014), but some clinical data and post-mortem studies show a decrease in astrocytic markers in alcohol-dependent populations that are thought to impact the integrity of the blood-brain barrier (Korbo, 1999; Miguel-Hidalgo et al., 2010). Few reports, however, have examined astrogliosis after binge-like consumption. In fact, this is the first report to indicate that non-dependent binge drinking increases GFAP immunoreactivity in the amygdala following the DID model (Figure 1). Our previous report found a similar response in the hippocampus with increased GFAP density after multiple cycles of DID ethanol exposure (Grifasi, McIntosh, et al., 2019). This finding agrees with the work of others who have shown that binge consumption in the DID model is associated with gene network expression changes that include astrocytic genes that regulate the neuroimmune response and glutamate synthesis (Mulligan et al., 2011). By using IHC, these studies were able to examine the subregions of the amygdala and found that the increase in GFAP density was only found in the BLA and not the CeA (Figure 1). Importantly, the change in GFAP immunoreactivity densitometric measures appear to be reflective of astrogliosis as there was no change in the number of astrocytes in either region. The regional specificity of alcohol-induced effects within the amygdala reflects the previous work showing that DID increased pro-inflammatory cytokines in the BLA but not the CeA (Marshall, Casachahua, et al., 2016; Marshall et al., 2017). The regional differences may reflect an increase susceptibility of the BLA to neuroimmune effects; however, it is important to denote that astrocyte morphological changes alone are not sufficient to fully understand the functional implications of astrocytes given their diverse morphological presentations (Khakh & Sofroniew, 2015; Oberheim et al., 2012). Regional differences in the effect of ethanol on GFAP immunoreactivity may also be associated with the basal expression of GFAP in the regions. The density of astrocytes in the CeA appeared lower than the BLA and may have impacted our ability to examine a significant effect of alcohol. At minimum, the change in GFAP densitometry shows that astrocytes are activated in a model of AUDs that does not elicit brain damage (Marshall, Casachahua, et al., 2016; Sprow et al., 2015).

Although the focus of this manuscript is on non-dependent binge drinking, it is important to acknowledge that astrogliosis from binge-like consumption in this relatively acute model of an AUD may indicate that the brain would be susceptible to further damage with secondary immunomodulators (Abbink et al., 2019; Saavedra et al., 2017). Neuroinflammatory priming due to alcohol misuse has been observed in other studies that focused on microglia- and alcohol-related brain damage (Barton et al., 2017; Marshall, Geil, et al., 2016; Zhao et al., 2013), but recent preclinical work indicates that early life exposure to alcohol in the DID model may prime astrocytes resulting in increased astrocytic reactivity and damage from traumatic brain injury (Mira et al., 2020). Recognizing the potential of binge drinking to make the brain more susceptible to damage is a critical consideration, especially as it relates to the various comorbidities of alcohol that also alter neuroimmune signaling including aging (Grifasi, Evans, et al., 2019; Norden et al., 2015; Perkins et al., 2019), infections (Qin et al., 2008; Singh et al., 2007; Vore et al., 2017), and traumatic brain injury (Karelina et al., 2018; Teng et al., 2015). Acknowledging the astrogliosis that occurs independent of brain damage and prior to evidence of dependence is critical in understanding the role of the neuroimmune system in AUDs.

One limitation of the current experiments is the inclusion of only male mice. Several laboratories have reported sex differences in neuroimmune responses and astrogliosis to pathological insults (Schwarz & Bilbo, 2012; Tronson & Collette, 2017). For example, Wilhelm’s et al. demonstrated increased GFAP+ cells and astrogliosis in the hippocampus in an alcohol dependence model. Importantly, this finding was only true of female but not male mice (Wilhelm et al., 2016). Astrocytic sexually dimorphic responses are especially relevant in the amygdala where some have reported that the non-pathological astrocyte number and morphology is different in males and females, with females having fewer and less complex astrocytes (Johnson et al., 2008, 2010). Additionally, sex differences have been reported in ethanol consumption in the DID which may have further influenced our findings (Crabbe et al., 2009; Sneddon et al., 2019). Future studies should determine whether sex is a critical biological variable in both astrogliosis and astrocytic mediated cellular signaling in non-dependent binge drinking.

4.2 |. GFAP Gq-signaling activation reduces ethanol consumption

Neuroimmunomodulation has repeatedly been shown to influence ethanol consumption with increased proinflammatory signaling inducing ethanol consumption (Agrawal et al., 2011; Blednov et al., 2012; Farokhnia et al., 2020), but this study specifically targeted astrocytes as a neuroimmunomodulatory cell. The data herein support the idea that astrocytes play an integral role in ethanol consumption (Adermark & Bowers, 2016). Critical to the interpretation of these data, DREADD appeared to have high fidelity to GFAP+ cells agreeing with the initial work of the McCarthy laboratory that characterized the GFAP-DREADD transgenic line (Agulhon et al., 2013; Otsu et al., 2019; Xie et al., 2017) (Figure S3). Astrocytic Gq-signaling activation decreased ethanol consumption below binge levels as evident by both peripheral CNO administration (Figure 2) and site-directed administration into the BLA (Figure 3). In the GFAP-hM3Dq transgenic mouse, peripheral CNO administration activates Gq signaling in GFAP+ cells throughout the nervous system (Agulhon et al., 2013; Xie et al., 2015, 2017). The effects of activation of GFAP-Gq signaling in these mice were not specific to ethanol and reduced both sucrose consumption and active mobility time, suggesting that reduced ethanol consumption was most likely due to impaired locomotor activity (Figures 2 and 4). Others have observed similar effects of GFAP-Gq-DREADD activation on locomotor activity (Agulhon et al., 2013). Future work should determine if lower peripheral CNO doses can be used that target maladaptive behaviors like binge drinking without impacting locomotor activity and general reward (Jendryka et al., 2019; MacLaren et al., 2016).

It is important to denote that CNO has been shown to reverse metabolize into clozapine in multiple species including mice (Gomez et al., 2017; Jendryka et al., 2019). Clozapine has been known to cross the blood–brain barrier and alter behavior. However, based on our data and the low concentration at which CNO was administered we suspect that there were no effects of clozapine. There were no behavioral differences between CNO and vehicle in either the littermates or WT mice in ethanol or sucrose consumption (Figure S2). This finding suggests that the CNO dose that may have metabolized to clozapine was negligible in our system.

Based on the IHC data herein and the non-specificity of our general GFAP-Gq-DREADD activation, we decided to examine the role of astrocytes specifically in the BLA by injecting CNO directly into the region. Our findings indicate that, unlike general activation, GFAP-Gq activation in the BLA only reduced ethanol consumption without directly affecting sucrose consumption, locomotor activity, or anxiety-like behavior (Figure 4). The singular effect of BLA Gq-astrocyte signaling on ethanol consumption suggest it may be a potential therapeutic target area and cell for AUD treatment. Amygdalar GFAP-DREADD activation has previously been shown to impact fear conditioning, but similar to the data herein, GFAP-DREADD activation did not impact basal anxiety measures (Martin-Fernandz et al., 2017). It is important to denote that our open field experiments were conducted in low light similar to homecages during the dark cycle. Additional lighting can act as a stressor further deterring mice from spending time in the center of the arena (Bouwknecht et al., 2007; Kulesskaya & Voikar, 2014). GFAP-Gq-DREADD activation has previously been shown to impact the motivation to consume alcohol when activated in the nucleus accumbens core (Bull et al., 2014), but this work highlights the importance of astrocytes in the amygdala and reinforces our work and others showing the influence of the neuroimmune system in alcohol-related behaviors (Bajo, Herman, et al., 2015; Bajo, Varodayan, et al., 2015; Doremus-Fitzwer et al., 2014; Gano et al., 2017; Marshall, Casachahua, et al., 2016; Marshall et al., 2017). The exact endogenous GPCR that may underlie the astrocytic role in alcohol consumption was not directly examined in these studies, but there are many candidates of GPCRs in the amygdala that influence ethanol consumption and are also found on astrocytes including metabotropic glutamate (Hwa et al., 2017; Joffe et al., 2018; Spampinato et al., 2018), neuropeptide Y (Cheunsuang & Morris, 2005; Hosli & Hosli, 1993; Robinson & Thiele, 2017; Schwarz et al., 2017), melanocortin (Caruso et al., 2013; Navarro, 2017), and corticotropin-releasing factor receptors (Chen et al., 2014; Lowery-Gionta et al., 2012; Rinker et al., 2017).

4.3 |. GFAP-Gq signaling ameliorates alcohol-induced decreased glutamate

There are a variety of mechanisms by which GPCR signaling in astrocytes may alter neuronal function and the behavioral measures observed herein. More extensive reviews on the potential effects of astrocytic GPCR signaling on neurotransmission have been previously published (Agulhon et al., 2012; Kofuji & Araque, 2020; Xie et al., 2015), but we chose to focus in on the role of astrocytes in glutamate regulation. Targeting glutamatergic systems has been suggested as a potential therapy for curbing alcohol misuse (Heilig & Egli, 2006; Rao et al., 2015), and these data indicate that astrocytes may be an important part of this response. Gq-signaling activation in GFAP+ cells was able to return the concentration of glutamate to control levels during intoxication (Figure 5). The ability of alcohol to reduce glutamatergic tone in the amygdala during intoxication while leading to an increased tone over time has been previously established (Hwa et al., 2017; Roberto et al., 2012), but this measure, albeit crude, supports that astrocytes can be manipulated to mitigate alcohol’s pharmacologic effects on glutamate. Unfortunately, the methodology used in these experiments does not allow for distinguishing specifically between neuronal and glial effects on glutamate nor was it feasible to separate the subregions of the amygdala. However, given the fidelity of the GFAP-DREADD construct, we hypothesize that astrocytic GPCR signaling regulation of glutamate may be one of the mechanisms responsible for the reduced ethanol consumption in the DID.

GFAP-DREADD activation has previously been shown to increase glutamatergic tone (Scofield et al., 2015), but it remains unclear if Gq-signaling activation directly stimulates gliotransmission (Fiacco & McCarthy, 2018; Savtchouk & Volterra, 2018). However, GFAP-Gq-DREADD activation may also alter glutamate concentration in the amygdala through mechanisms like glutamate transporter localization or glutamate metabolism (Nijboer et al., 2013; Wetherington et al., 2008; Xie et al., 2015). Regardless of the internal mechanism, astrocytic control of glutamate would alter neurotransmission. Astrocytic control of glutamate represents a viable target that can be manipulated to curb alcohol intake. In fact, the work of the Bechtholt laboratory indicated that amygdalar astrocyte regulation of glutamate uptake via GLT-1 could reduce ethanol consumption (Smith et al., 2014). The influence of GLT-1 in the amygdala on addiction-like behaviors and anxiety further suggests that astrocytic regulation of glutamate is a potential target for AUDs (John et al., 2015; Scofield & Kalivas, 2014). Our findings add to this body of work and suggest that astrocytic GPCR signaling may represent a novel target for controlling glutamatergic tone and therein curb alcohol abuse.

5 |. CONCLUSIONS

Binge-like alcohol consumption and its reciprocal relationship with the neuroimmune system has been a hot topic in the AUD field. Our current findings indicate that non-dependent binge-alcohol consumption increases GFAP immunoreactivity in the BLA. Moreover, GFAP-Gq-DREADD activation decreases alcohol consumption, but general Gq-DREADD activation in astrocytes had more broad effects compared with localized activation in the BLA. It appears that the effects of alcohol on astrocytes are more prevalent in the BLA and that the region represents a crucial hub for ethanol consumption related behaviors. Our initial work suggests that this may be mediated by the astrocytic influence on glutamate regulation, but much more work would need to be done to truly distinguish the downstream signaling of GFAP-Gq-receptor activation to directly link it to alcohol consumption.

Supplementary Material

Supplementary Tables and Figures

TABLE S1 Active time in open field test

FIGURE S1 Simple linear regressions indicated that there was not a significant correlation between average consumption and GFAP immunoreactivity (a), but BECs were significantly, positively correlated with the GFAP immunoreactivity in the BLA (b)

FIGURE S2 Vehicle administration peripherally had no effects on ethanol consumption (a) or related BECs (b) suggesting that the genotypic differences do not cause innate changes in ethanol related behaviors. Moreover, a direct comparison of CNO versus vehicle (Test 2 vs. Test 3) in the littermate and WT animals were not significantly different suggesting that CNO metabolism did not significantly impact ethanol consumption

FIGURE S3 Photomicrographs of the BLA (a–e) and hippocampus (f–j) were obtained with a 10× objective from GFAP-DREADD transgenic animals. GFAP was tagged with Alexa Fluor ® 488 (a,f), mCherry was tagged with Alexa Fluor ® 568 (b,g), and DAPI (d,i) was used to visualize all nuclei. An overlay of one of the serial sections of both the BLA (e) and the hippocampus (j) show that the mCherry associated with the DREADD expression labels the bushy exterior of astrocytes while the GFAP is only expressed within the filaments. A 3-D rendering of both the BLA (c) and the hippocampus (h) further illustrate the expression pattern. Importantly nuclei without GFAP did not appear to have the DREADD construct. White arrows in panels e and j show DREADD on the membrane surface of GFAP+ cells. Scale bars on 2D images = 40 μm. The 3D rendering has both and x and y scale in μm

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Significance.

This study provided evidence that non-dependent binge-like drinking impacts astrogliosis. Importantly, the relationship is bidirectional, and activation of Gq signaling in astrocytes reduced binge-like consumption of ethanol. This study revealed that the astrocytes of the basolateral amygdala are of particular interest and have a more discrete effect on ethanol consumption compared with other rewards.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.