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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Neuropharmacology. 2021 Sep 21;200:108795. doi: 10.1016/j.neuropharm.2021.108795

Vesicular glutamate transporter 2-containing neurons of the centrally-projecting Edinger-Westphal nucleus regulate alcohol drinking and body temperature

Alfredo Zuniga 1,*, Monique L Smith 1, Maya Caruso 1, Andrey E Ryabinin 1
PMCID: PMC8694014  NIHMSID: NIHMS1744660  PMID: 34555367

Abstract

Previous studies in rodents have repeatedly demonstrated that the centrally-projecting Edinger-Westphal nucleus (EWcp) is highly sensitive to alcohol and is also involved in regulating alcohol intake and body temperature. Historically, the EWcp has been known as the main site of Urocortin 1 (Ucn1) expression, a corticotropin-releasing factor-related peptide, in the brain. However, the EWcp also contains other populations of neurons, including neurons that express the vesicular glutamate transporter 2 (Vglut2). Here we transduced the EWcp with adeno-associated viruses (AAVs) encoding Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the EWcp in alcohol drinking and in the regulation of body temperature. Activation of the EWcp with excitatory DREADDs inhibited alcohol intake in a 2-bottle choice procedure in male C57BL/6J mice, whereas inhibition of the EWcp with DREADDs had no effect. Surprisingly, analysis of DREADD expression indicated Ucn1-containing neurons of the EWcp did not express DREADDs. In contrast, AAVs transduced non-Ucn1-containing EWcp neurons. Subsequent experiments showed that the inhibitory effect of EWcp activation on alcohol intake was also present in male Ucn1 KO mice, suggesting that a Ucn1-devoid population of EWcp regulates alcohol intake. A final set of chemogenetic experiments showed that activation of Vglut2-expressing EWcp neurons inhibited alcohol intake and induced hypothermia in male and female mice. These studies expand on previous literature by indicating that a glutamatergic, Ucn1-devoid subpopulation of the EWcp regulates alcohol consumption and body temperature.

Keywords: alcohol, glutamate, urocortin, centrally-projecting Edinger Westphal, designer receptors exclusively activated by designer drugs, thermoregulation

1. Introduction

Alcohol use disorder (AUD) is a progressive disease characterized by compulsive alcohol consumption with detrimental consequences for both the affected individual and society as a whole. AUD is observed in an estimated 16 million people in the United states (SAMHDA, 2017). Globally, 3.3 million people die from alcohol-related causes annually, making it the fifth leading cause of premature death and disability (Lim et al., 2012). Studies on the transition into compulsive alcohol use have identified neuroadaptations within several neurotransmitter and neuromodulatory systems, including plasticity within GABAergic, glutamatergic, dopaminergic and peptidergic neurons (Koob, 2014).

Whole brain mapping approaches have been employed to identify brain regions and neuronal populations preferentially sensitive to alcohol. Among them, changes in expression of immediate early genes, such as c-Fos, have been extensively mapped across rodent brains after both voluntary (i.e., self-administration) and involuntary (i.e., administration by an experimenter) modes of alcohol (ethanol- ETOH) exposure. While experimenter-administered ETOH induced c-Fos in a large number of brain regions (Ryabinin et al., 1995, 1997; Ryabinin & Wang, 1998; Thiele et al., 1997), voluntary alcohol drinking-induced c-Fos in a much more circumscribed number of neuronal populations. Specifically, in the earliest study in rats, consumption of ETOH-supplemented beer induced c-Fos only in the Edinger-Westphal nucleus (EW) (Topple et al., 1998). The EW was also the only brain region showing c-Fos induction in rats self-administering ETOH in an operant procedure (Weitemier, Woerner, Backstrom, Hyytia, & Ryabinin, 2001). The induction of c-Fos in the EW in these studies was ETOH-specific as no induction was observed following consumption of non-alcoholic beer or saccharine. While multiple studies performed in mice showed additional brain regions that also showed increased c-Fos induction following ETOH drinking, such as the central nucleus of amygdala (CeA) and nucleus accumbens (NAcc), the induction of c-Fos in the EW following voluntary ETOH consumption was a highly consistent finding, demonstrating preferential sensitivity of the EW to self-administered ETOH (Bachtell, Wang, Freeman, Risinger, & Ryabinin, 1999; Ryabinin, Bachtell, Freeman, & Risinger, 2001; Ryabinin, Wang, Freeman, & Risinger, 1999; Ryabinin, Galvan-Rosas, Bachtell, & Risinger, 2003). Further confirming this idea, the induction of c-Fos in the EW was positively correlated with the amount of consumed ETOH (Giardino et al., 2017; Sharpe et al., 2005).

The EW was traditionally considered as an area involved in oculomotor adaptation (Edinger, 1885; Westphal, 1887). However, early studies raised doubts about this (Cajal, 1995), and more recent studies have revised this classic idea (Kozicz et al., 2011). Specifically, the EW is currently understood to consist of two main subregions: the preganglionic EW (EWpg), a cholinergic region primarily involved in oculomotor functions, and the centrally-projecting EW (EWcp), a region enriched in neuropeptides that projects to several central sites (Cavani et al., 2003; May, Reiner, & Ryabinin, 2008; Ryabinin, Tsivkovskaia, & Ryabinin, 2005; Weitemier, Tsivkovskaia, & Ryabinin, 2005). Urocortin 1 (Ucn1), a peptide of the corticotropin-releasing factor (CRF) family is preferentially expressed in the EWcp (Kozicz et al., 2011; Vaughan et al., 1995; Weitemier et al., 2005). Consequently, Ucn1 can serve as a reliable marker to identify EWcp neurons. C-Fos mapping studies have subsequently confirmed that experimenter- and self-administered ETOH specifically activates Ucn1-containing neurons (Bachtell et al., 2002a; Ryabinin et al., 2003; Spangler et al., 2009).

Subsequent studies indicated that the EWcp is not only sensitive to ETOH, but is also involved in the regulation of ETOH-seeking behaviors. Genetic studies in mice and rats selectively-bred for differences in alcohol-related behaviors showed consistently higher levels of Ucn1 expression in alcohol-preferring versus alcohol non-preferring rodent strains (Bachtell et al., 2002a; Kiianmaa et al., 2003; Turek et al., 2005), as well as in mouse strains selected for higher ETOH-induced hypothermia (Bachtell et al., 2002a). Electrolytic lesions of the EWcp decreased ETOH consumption and attenuated ETOH-induced hypothermia (Bachtell et al., 2004; Weitemier & Ryabinin, 2005). Ucn1 knockout mice have been shown to consume less alcohol than their wildtype littermates across several, but not all, paradigms and showed an attenuated conditioned place preference for ethanol (Giardino et al., 2011). The decreased ETOH intake in Ucn1 knockout mice is in part dependent on an intact EWcp (Giardino et al., 2011). Knockdown of Ucn1 in the EWcp using a lentiviral shRNA approach also decreased escalated ETOH intake in mice (Giardino et al., 2017). Our more recent studies described that direct pharmacologic inhibition of the EWcp did not affect conditioned place preference for ethanol, but increased body temperature (Zuniga, Ryabinin, & Cunningham, 2020).

The accumulated evidence for the EWcp’s involvement in the regulation of ETOH-related behaviors indicates the need for future investigation of its function. In addition, it is increasingly recognized that the EWcp is not a uniform structure but rather consists of multiple cellular subpopulations (Zuniga & Ryabinin, 2020). Specifically, while the predominant population of neurons within the EWcp co-expresses peptides Ucn1, cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1(Kozicz, 2003; Okere et al., 2010), a smaller subpopulation expresses tyrosine hydroxylase (Bachtell et al., 2002b), whereas a third subpopulation co-express cholecystokinin (CCK) and substance P (Innis & Aghajanian, 1986). Our own analysis of the Allen Brain Atlas (Lein et al., 2007) also suggested the presence of Vglut2-positive neurons within the EWcp. However, it is not clear whether or not these Vglut-2 neurons are separate from the glutamatergic population recently identified in the vicinity of the EWcp (Zhang et al., 2019).

The current study aimed at characterizing the contribution of specific subpopulations of EWcp neurons to the regulation of ETOH intake. Using chemogenetic approaches we first modulated activity of all neurons within the EWcp during alcohol drinking. Subsequent experiments indicated that the EWcp neurons inhibiting ETOH consumption are not Ucn1-, but rather Vglut2-positive. A further chemogenetic experiment showed that these Vglut2 neurons also regulate body temperature.

2. Materials and Methods

2.1. Animals

For Experiment 1, adult male C57BL/6J (B6) mice (The Jackson Laboratory, CA, 7–8 weeks upon arrival) were housed three-five per cage upon arrival and allowed to acclimate to the animal colony for seven days prior to surgery. Male Ucn1 WT and KO mice used for Experiment 2 were littermates from heterozygous matings. Ucn1 KO mice contained a deletion of exon 2 of the Ucn1 gene and were originally on a 12961/SvJ × B6 background (Vetter et al., 2002) and were backcrossed for over 15 generations onto B6 background. For Experiment 3–4, male and female Vglut2-Cre and WT littermates were generated from Vglut2-ires-Cre knock-in on a B6 background (Jackson Labs stock # 028863) and WT breeding pairs purchased from the Jackson Laboratory. Following acclimation to the animal colony (for Experiment 1) or after reaching adulthood (Exp 2–4), all mice were subjected to stereotaxic surgery (described below), individually housed, and allowed to recover for 7–10 days prior to the start of the experiment. Following recovery, mice were transferred to the experimental room with a 12/12 reverse light/dark cycle (lights on at 9:00 PM) for a 7-day acclimation period prior to the initiation of the experiments. All housing rooms were temperature and humidity controlled and food and water were available ad libitum. All protocols were approved by the Oregon Health & Science University animal care and use committee and performed within the National Institutes for Health Guidelines for the Care and Use of Laboratory Animals, as well as the Guidelines for the Care and use of Mammals and Behavioral Research.

2.2. Drugs

ETOH solutions were prepared in tap water from 95% ethyl alcohol. CNO (1 mg/kg, Sigma-Aldrich, MO) was dissolved in 0.5% DMSO (Sigma-Aldrich, MO) in sterile saline.

2.3. Stereotaxic surgery and viral infusions

Seven to ten days prior to the start of each experiment, mice were transported to a suite for stereotactic surgery. Mice were anesthetized via 5% isoflurane delivered in oxygen via a precision vaporizer (DatexOhmeda, WI). Following induction, mice were maintained under 1–2% isoflurane anesthesia and secured in a stereotaxic frame. Once in the stereotaxic frame, mice were given a subcutaneous injection of the non-steroidal anti-inflammatory drug carprofen (5 mg/kg) to minimize pain and discomfort. The EWcp (A/P −3.45 mm from bregma, M/L −1.20 mm, D/V −3.5 mm) was targeted at a 20° angle (to avoid the sagittal sinus) based on previous studies. A single burr hole was drilled 3.45 mm from bregma, and 1.2 mm from the midline, and a stainless-steel injector (32 ga) attached to a 1 μl Hamilton syringe via polyethylene tubing was lowered above the EWcp. Mice in Experiment 1 received 150 nL of the adeno-associated (serotype 8) excitatory AAV8-hSyn-hM3D-Gq-mCherry (hM3Dq), inhibitory AAV8-hSyn-hM4D-Gi-mCherry (hM4Di) or control AAV8-hSyn-GFP (GFP) DREADD viruses (Addgene, MA). All mice in Experiment 2 received 150 nL of the excitatory (hM3Dq) virus, while all mice in Experiment 3 received 150 nL of the Cre-dependent AAV8-hSyn-DIO-HA-hM3D-Gq-IRES-mCitrine (DIO-hM3Dq) virus. Finally, mice in Experiment 4 received either the Cre-dependent AAV8-hSyn-DIO-HA-hM3D-Gq-IRES-mCitrine (DIO-hM3Dq), or the control AA8-hSyn-GFP (GFP) virus. In all 4 experiments viral injections were conducted over the course of 5 min. Injectors were left in place for 10 min and extracted over the course of 5 min. For all experiments, injections were considered a “hit” when neuronal expression of the virus was limited to < 1.0 mm total diameter including/surrounding the EWcp. When spread of the virus was beyond this area, it was considered a “miss”. This procedure led to an 85% success rate. Any misses were excluded from analysis.

2.4. 24-h access 2-Bottle Choice (2-BC)

Unless otherwise specified, all mice in Experiment 1–3 were exposed to a 12-day 24-h access 2-bottle choice following recovery from surgery and acclimation to single-housing. During the acclimation period, mice were allowed 24-h access to two 25ml glass cylinder bottles with metal sipper tubes (containing water) on either side of the cage, with food evenly spread across the cage top. Throughout the ethanol drinking period, mice received 24-h access to two bottles: one containing water and one containing increasing concentrations of ETOH (3–10%) dissolved in water. Each concentration was available for 4 days. All bottles were introduced and fluid levels were recorded on a daily basis at 2 h into the dark cycle. The locations of the bottles on the cages (left vs. right) were alternated daily to avoid the potential confound of an inherent side preference.

2.5. Experiment 1: Effect of chemogenetic modulation of the EWcp on ETOH Intake

Experiment 1 was conducted to examine the effects of chemogenetic inhibition or activation on ETOH intake. As described above, all mice received a stereotaxic infusion of an AAV carrying either the hM4Di, hM3Dq, or the GFP-control DREADD. A pilot experiment was first conducted to test whether CNO or vehicle injection affected levels of c-Fos immunoreactivity in mice transfected with the hM4Di or hM3Dq DREADD. In this initial pilot experiment, after recovering from surgery, a subset of mice were given a single injection of CNO (1 mg/kg i.p.) or vehicle and were sacrificed via CO2 inhalation 2 h later. All other mice received a single i.p. injection of CNO or vehicle at the beginning of the dark cycle on day 13, following 12 days of 24 h access to ETOH and water. Treatment groups were counterbalanced based on ETOH intake during the 12 days of drinking. Following the i.p. injection of CNO or vehicle, mice were given access to two bottles, one containing 10% ETOH, and the other water, and intake was measured at 4 h post injection.

2.6. Experiment 2: Effect of chemogenetic activation of the EWcp on ETOH intake in Ucn1 WT and KO Mice

Experiment 2 was designed and carried out in order to characterize the involvement of Ucn1 in hM3Dq-mediated changes in ETOH intake. As such, male Ucn1 WT and KO mice were all transfected with the hM3Dq DREADD. As with Experiment 1, mice in Experiment 2 received a single i.p. injection of CNO or vehicle at the beginning of the dark cycle on day 13, following 12 days of 24 h access to ETOH and water. Within WT and Ucn1 KO mice, treatment groups were counterbalanced based on ETOH intake during the 12 days of drinking. Following the i.p. injection of CNO or vehicle, mice were given access to two bottles, one containing 10% ETOH, and the other water, and intake was measured at 4 h post injection.

2.7. Experiment 3: Effects of chemogenetic activation of Vglut2-expressing EWcp neurons on ETOH Intake

Using a Cre-dependent DIO-hM3Dq DREADD in male and female Vglut2-Cre and WT mice, Experiment 3 examined the role of EWcp Vglut2-expressing neurons in the modulation of ETOH intake. For Experiment 3, all mice were given both vehicle and CNO i.p. injections in a counter-balanced manner, spread 48 h apart. Specifically, all mice were given an i.p. injection of CNO or vehicle at the beginning of the dark cycle on day 13, following 12 days of 24 h access to ETOH and water. Intake was then measured 2 h, 4 h, and 24 h after the i.p. injection. Following an additional 24 h “wash-out”, during which mice had continued access to both ETOH and water, mice were given a second i.p. injection of the treatment opposite of what they initially received, at the beginning of the dark cycle. Again, intake was measured 2 h, 4 h, and 24 h after the injection. Mice were then given an additional seven day “wash-out” period during which they only had access to water. Following this wash out, mice were given an i.p. injection of CNO or vehicle at the beginning of the dark cycle on the 8th day, and were then given access to 2 bottles, one containing 2% sucrose, and one containing water for 2 h during which intake was measured. After the 2 h period, mice were again given access to only water. Forty-eight hours later, mice were given a second injection (opposite of what they received the 1st sucrose day), and again had access to 2% sucrose and water for 2 h.

2.8. Experiment 4: Effect of activation of Vglut2-expressing neurons in the EWcp on body temperature.

Experiment 4 was conducted to investigate the effect of chemogenetic activation of Vglut2-expressing neurons in the EWcp on body temperature. Male and female Vglut2-Cre mice received a stereotaxic microinjection of an AAV expressing either the Cre-dependent excitatory DREADD (DIO-hM3Dq) or a control green fluorescent protein (GFP) targeted at the EWcp. Following recovery from surgery, mice were handled and habituated to the temperature probe once a day, for three days. Following habituation, baseline rectal temperatures were assessed, immediately followed by an i.p. injection of either CNO or vehicle. Rectal temperatures were then measured 30, 45, and 60 min after the CNO injection. This timeline for temperature readings is based on previous work in our lab that has shown that EWcp manipulations can alter body temperature in the immediate 60–90 minutes after the manipulation (Bachtell et al., 2004, Zuniga et al., 2020). Seven days after the initial experimental injection, baseline rectal temperatures were again measured and mice were given an i.p. injection of the opposite treatment that they received previously. If mice received CNO initially, they received vehicle the second time, and vice versa. Again, rectal temperatures were measured 30, 45, and 60 min after the injection.

2.9. Brain Extraction and Immunohistochemistry

On the final day of each experiment, mice were sacrificed via CO2 inhalation, brains were extracted, post-fixed for 24 hours in 2% paraformaldehyde/phosphate-buffered saline (PBS) and cryopreserved in 20–30% sucrose/PBS. Brains were sliced at 30 μm and processed for Ucn1, mCherry, mCitrine, HA, Vglut2, and c-Fos immunohistochemistry. The subset of mice used for the c-Fos analysis in Experiment 1 were sacrificed 2 h after they received an injection of CNO or vehicle. Unless noted otherwise, all steps were performed in 0.3% Triton-X/Tris-buffered saline (TBS) and preceded by three washes in TBS. The sections were rinsed for 30 min in 1% sodium borohydride in TBS, and blocked in 5% normal donkey serum (Jackson Laboratories) for 45 min. The tissue was then incubated with 1:5000 goat polyclonal Ucn1 antibody (Santa Cruz), 1:15000 rabbit polyclonal c-Fos antibody (Sigma-Aldrich, MO), 1:1000 rabbit polyclonal influenza hemagglutinin (HA) antibody, 1:850 rabbit or goat polyclonal green fluorescent protein antibody (for mCitrine detection) (Abcam, MA), 1:850 rabbit polyclonal Vglut2 antibody (Synaptic Systems, Germany), or 1:2500 rabbit polyclonal DS-Red (for mCherry detection) (Clontech, CA). This was followed by 1 h incubations with AlexaFluor 555-labeled and AlexaFluor 488-labeled secondary antibodies (raised in donkey) (Invitrogen Thermo Fisher, MA). Finally, slices were washed with PBS, mounted on gelatinized slide and coverslipped with Prolong Gold (Invitrogen Thermo Fisher, MA). Co-localization of immunoreactivity was quantified manually using a Leica DM4000 microscope and ImageJ software.

2.10. Statistics

Based on the appropriate concentration, ETOH consumption in mL was converted to grams and divided by the animal’s body weight to give daily intake scores expressed in grams per kilogram (g/kg). Sucrose and water consumption were divided by the animal’s body weight to give values expressed in mL/kg. The initial 12-day drinking data were averaged over the 4 days of access to each concentration, such that a single value for 3, 6, and 10% were calculated for each animal. These data were then analyzed via unpaired t-test or ANOVAs for each concentration, where appropriate. On treatment days for Experiment 1 and 2, drinking data were analyzed via a two-way ANOVA with Virus and Treatment (Experiment 1) or Genotype and Treatment (Experiment 2) as the between group factors. Bonferroni post-hoc analyses were conducted when appropriate. For Experiment 3, intake data following treatment were analyzed via a two-way repeated measures (RM) ANOVA with Treatment as the within groups factor, and Genotype as the between groups factor. For Experiment 4, body temperate temperature measurements were converted into a change-from-baseline value, and these data were analyzed using a three-way RM ANOVA with Time and Treatment as within-subject factors and Virus as a between-subject factor. For c-Fos cell-count analyses, a single c-Fos value was calculated for each animal by averaging the cell counts across the 5–7 sections that encompassed the EWcp in each animal. Thus, each animal had a single c-Fos cell count value for the EWcp. C-Fos data were analyzed using a two-way ANOVA with Virus (hM3Dq vs hM4Di) and Treatment (CNO vs vehicle) as the between group factors. For the colocalization analysis performed for Exp. 1, 3–4 sections encompassing the EWcp were counted for 4 mice. The total number of mCherry- and Ucn1-positive cells for each section were counted an averaged across mice. All significant statistical findings and their reports are described in the main text. Statistical analyses that did not reach significance are all reported in Supplementary Table #1.

3. Results

3.1. Experiment 1: Effect of chemogenetic modulation of the EWcp on ETOH Intake

To investigate the effect of chemogenetic modulation of the EWcp on ETOH intake, we transfected male C57BL/6J mice with either the GFP, hM4Di or hM3Dq DREADD (Fig. 1A). Surprisingly, although we observed robust DREADD expression (as indicated by mCherry expression) in the EWcp (Fig. 1B), there were no instances of mCherry expression in Ucn1-expressing neurons, suggesting that this AAV serotype (AAV8) did not transfect Ucn1-neurons in the EWcp (Fig 1C). Similarly, immunohistochemical imaging for the neuropeptide CART, which is heavily colocalized with Ucn1 in the EWcp (Xu et al., 2009), also revealed no overlap between the CART-expressing neurons and those that expressed mCherry (Supplementary Fig. 2A–D). In order to assess the effects of DREADD activation on transfected neurons in the EWcp, a subset of ETOH-naïve mice were given a CNO or vehicle injection and were sacrificed 2h post injection for c-Fos analyses. As expected, a single injection of 1.0 mg/kg CNO (but not vehicle) led a significant increase in c-Fos-positive cells in the EWcp of mice that had received the hM3Dq DREADD, but not the hM4Di DREADD (Supplementary Fig. 1B). In support of this, a two-way ANOVA for the average number of c-Fos-positive cells in the EWcp detected a main effect of Virus [F(1, 15) = 30.72, p = 0.0001] and Treatment [F(1, 15) = 23.23, p = 0.0001] and a significant Virus × Treatment interaction [F(1, 15) = 29.25, p = 0.0001]. The Bonferroni-adjusted post-hoc analysis confirmed a significant increase in c-Fos induction in mice that were transfected with the hM3Dq DREADD (p < 0.0001).

Fig. 1. Chemogenetic activation of the EWcp attenuates 10% ETOH intake.

Fig. 1.

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Following recovery from surgery and acclimation, mice transduced with either the GFP-control, hM4Di, or hM3Dq DREADD were allowed access to increasing concentrations of ETOH (3–10%) prior to treatment with CNO or vehicle. A) Experimental timeline. B) Representative immunofluorescent image taken at 2.5X magnification of hM3Dq expression in the EWcp, scale bar denotes 250μm, dashed box denotes area imaged at higher magnification (10X, scale bar inside dashed box denotes 50μm) C) Representative immunofluorescent image taken at 20X magnification of mCherry (red) and Ucn1 (green) expression in the EWcp, denotes 25μm. When quantified (bottom right panel), no instances of mCherry and Ucn1 co-expression were observed. D) No differences in 24-h ETOH (g/kg ETOH) intake between mice transduced with the GFP, hM4Di or hM3Dq viruses were detected at any of the ETOH concentrations. E) On the fifth day of 10% ETOH access, an injection of CNO (1.0 mg/kg) led to a significant decrease in ETOH intake in hM3Dq-transduced mice compared to vehicle-treated mice. No significant differences in intake were detected in GFP- or hM4Di-transduced mice. F) There were no differences in water intake in any group following injection with CNO or vehicle. **; significant difference in intake between groups according to Bonferroni adjusted post-hoc, p < 0.01.

Over the course of the 12-day baseline drinking period, there were no differences in ETOH intake based on the type of virus mice were transfected with (Fig. 1D), regardless of the ETOH concentration mice were drinking. Following the 12 days of 24h 2-BC access, mice were given a single i.p. injection of either CNO or vehicle, and ETOH intake was measured 4h after. Activation of the EWcp in hM3Dq-transfected mice via an injection of 1.0 mg/kg CNO significantly decreased 4h ETOH intake on day 13 (Fig. 1E). CNO did not alter ETOH intake in GFP- or hM4Di-transfected mice. Lastly, CNO did not alter water intake in any of the viral groups during the 4h period (Fig. 1F). In support of these observations, the two-way ANOVA for ETOH intake 4h-post injection detected a main effect of treatment [F(1, 24) = 4.9, p = 0.036, as well as a significant Virus × Treatment interaction [F(2, 24) = 4.7, p = 0.018]. A Bonferroni-adjusted post-hoc analysis found that there was significantly lower ETOH intake in CNO- versus Vehicle-treated mice that were transfected with the hM3Dq DREADD, but not in mice that were transfected with the hM4Di or GFP DREADDs (p = 0.003). Lastly, a main effect of Virus was detected for the water intake 4h post injection [F(2, 24) = 4.1, p = 0.028]. No other significant effects or interactions were detected.

3.2. Experiment 2: Effect of chemogenetic activation of the EWcp on ETOH intake in Ucn1 WT and KO Mice

Based on the finding above showing that chemogenetic activation of non-Ucn1 expressing neurons in the EWcp decreased ETOH intake, we next tested if Ucn1 expression was required for this hM3Dq-mediated decrease in intake by using Ucn1 KO and Ucn1 WT mice. Average ETOH over the course of the 12-day 2BC procedure did not significantly differ between Ucn1 WT and KO mice, at any of the ETOH concentrations (Fig 2B). Activation of the EWcp via an injection of CNO significantly decreased 10% ETOH intake in both and WT and Ucn1 KO mice 4h post injection, compared to mice that received a vehicle injection (Fig. 2C). In contrast, CNO did not affect water intake in either genotype (Fig. 2D). A two-way ANOVA for ETOH intake post-treatment found a main effect of Treatment [F(1,16) = 11.6, p = 0.003], confirming that CNO decreased ETOH intake in both genotypes. A main effect of Genotype that was trending towards significance [F(1, 16) = 3.62, p = 0.075) was also detected, indicating that although ETOH intake between genotypes was not significantly different, Ucn1 WT mice did tend to drink more during the 4h period. No other significant main effects or interactions were detected.

Fig. 2. Chemogenetic activation of the EWcp inhibits 10% intake in WT and Ucn1 KO mice.

Fig. 2.

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Following recovery from stereotaxic surgery for hM3Dq delivery into the EWcp, WT and Ucn1 KO mice were allowed access to increasing concentrations of ETOH (3–10%) prior to treatment with CNO or vehicle. A) Experimental timeline. B) g/kg ETOH intake did not significantly differ at any concentration during the 12-day drinking period when comparing WT and KO mice. C) Injection of CNO (1.0 mg/kg) significantly attenuated g/kg ETOH intake in both WT and Ucn1 mice. D) CNO did not alter water intake in either Ucn1 WT or KO mice. ##; main effect of treatment, p < 0.01.

3.3. Experiment 3: Effects of chemogenetic activation of Vglut2-expressing EWcp neurons on ETOH Intake

Following experiments 1 and 2, which demonstrated that activation of non-Ucn1-expressing cells in the EWcp decrease ETOH intake, we next sought to identify the neurochemical nature of the non-Ucn1-expressing neurons transfected by AAV8 viruses in the EWcp. Our search of the Allen Brain Atlas (Lein et al., 2007) indicated the presence of vGlut2 mRNA in the vicinity of the EWcp, suggesting that these neurons are glutamatergic. Thus, we transfected the EWcp of Vglut2-cre mice and their WT littermates with an AAV8 expressing the Cre-dependent excitatory DREADD (DIO-hM3Dq). Robust expression of the DIO-hM3Dq DREADD (as determined by mCitrine expression) was observed in the EWcp of Vglut2-Cre mice but not WT mice (Fig 3B–C). Subsequent fluorescent immunohistochemistry for the hM3Dq-fused influenza hemagglutinin (HA) tag, a marker of receptor expression, and for Ucn1, demonstrated that Ucn1-positive neurons did not express the Cre-dependent hM3Dq DREADD (Supplementary Fig. 3A–C), further indicating that Ucn1- and Vglut2-expressing neurons are two distinct populations of neurons in the EWcp.

Fig. 3. Chemogenetic activation of EWcp glutamatergic neurons decreases 10% ETOH intake.

Fig. 3.

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Following stereotaxic surgery and recovery, WT and Vglut2-Cre mice transduced with DIO-hM3Dq were allowed access to increasing concentrations of ETOH (3–10%) prior to treatment with CNO or vehicle. A) Experimental timeline. B, C) Representative photomicrograph taken at 2.5X magnification illustrating expression of DIO-hM3Dq in the EWcp of Vglut2-Cre but not WT mice, scale bar denotes 250μm, dashed box denotes area imaged at higher magnification (10X, scale bar inside dashed box denotes 50μm). D) Injection of CNO (1.0 mg/kg, i.p.) significantly attenuated g/kg ETOH intake during the 2BC test in Vglut2-Cre but not WT mice. E-G) Injection of CNO (1.0 mg/kg) did not alter water or sucrose intake during the ETOH and sucrose 2BC tests in WT or in Vglut2-Cre mice. ****; significant difference in intake between treatments according to Bonferroni adjusted post-hoc, p < 0.0001.

Throughout the 12-day 2BC drinking period there were no genotype-dependent differences in ETOH intake, indicating that Cre-expression did not alter baseline drinking behavior (Supplementary Fig. 4A). When ETOH intake was measured 2hrs after treatment of CNO or vehicle, only Vglut2-Cre mice displayed a strong reduction in ETOH intake when treated with CNO (Fig. 3D). This decrease was specific to ETOH, as CNO treatment did not alter water intake (Fig 3E). Additionally, CNO had no effect on sucrose or water intake during a sucrose intake test (Fig. 3F–G). In support of these observations, a two-way RM ANOVA (Treatment × Genotype) for ETOH intake 2h-post treatment revealed a main effect of Genotype [F(1, 36) = 12.4, p = 0.001] and of Treatment [F(1, 36) = 32.9, p = 0.0001], and importantly a significant Genotype × Treatment interaction [F(1, 36) = 13.9, p = 0.0007]. To better understand this significant interaction, we conducted a Bonferroni adjusted post-hoc. This post-hoc revealed that only Vglut2-Cre mice displayed a significant decrease in ETOH intake following CNO administration (p = 0.0001). Lastly, two-way RM ANOVAs (Treatment × Genotype) for water intake revealed main effects of genotype, indicating that Vglut2-Cre mice drank significantly less water than their WT littermates during the 2hr period when ETOH and water were provided [Fig. 3E, F(1, 36) = 5.6, p = 0.023], as well as during the sucrose intake test [Fig. 3G, F(1, 25) = 9.05, p = 0.006].

When intake was measured again 4hrs after CNO injections, the decrease in intake observed in Vglut2-Cre mice given CNO were no longer present, indicating that hM3Dq-mediated activation of glutamatergic neurons in the EWcp only decreased intake during the first 2hrs-post injection (Supplementary Fig. 4B). Similarly, at the 4hr timepoint, there were no differences in water intake between genotypes and treatments (Supplementary Fig. 4C). In support of these findings, a two-way RM ANOVA (Treatment × Genotype) for ETOH intake 4hrs-post treatment revealed no significant main effects of Treatment [F(1, 36) = 3.253, p = 0.079], Genotype [F(1, 36) = 0.501, p = 0.484], nor a Genotype × Treatment interaction [F(1, 36) = 0.682, p = 0.414]. Likewise, a two-way RM ANOVA (Treatment × Genotype) for water intake 4hr-post treatment revealed no significant main effects (Treatment: [F(1, 36) = 1.047, p = 0.313], Genotype: [F(1, 36) = 3.063, p = 0.089]), nor a significant Genotype × Treatment interaction [F(1,36) = 0.442, p = 0.511]

3.4. Experiment 4: Effect of activation of Vglut2-expressing neurons in the EWcp on body temperature.

Previous studies had demonstrated the involvement of the EWcp in thermoregulation, particularly through Ucn1-mediated mechanisms (Bachtell et al., 2002; Telegdy et al., 2006). However, the potential involvement of additional neurons in the EWcp in thermoregulation had not been addressed. Therefore, we also used a Cre-dependent hM3Dq DREADD to activate Vglut2-expressing neurons in the EWcp and determine the involvement of these neurons in this physiological process. We observed no baseline temperature differences between virus or treatment groups, and thus calculated the mean change from baseline temperature during the 60 mins following an injection of either CNO or vehicle. Following the injection of either CNO or vehicle, body temperatures fluctuated varyingly over the course of the 60 min (Fig. 4B). In mice that were transfected with the hM3Dq DREADD however, CNO-mediated activation of Vglut2-expressing neurons in the EWcp resulted in the largest change in core body temperature, as evidenced by a sharp decrease over time. A three-way RM ANOVA with Time and Treatment as within-subject factors and Virus as a between-subject factor revealed main effects of Time [F(3, 72) = 14.9, p < 0.0001], Treatment [F(1,24) = 19.7, p < 0.0001] and virus [F(1,24) = 6.73, p = 0.016]. Additionally, significant Treatment by Virus [F(1, 24) = 9.08, p = 0.006] and Treatment by Time [F(3, 72) = 10.8, p < 0.0001] interactions were detected, as well as a significant 3-way Time by Treatment by Virus interaction [F(3,72) = 8.3, p < 0.0001]. In order to better understand this 3-way interaction, a separate two-way RM ANOVA (Treatment, Virus) for the change in body temperature for the 60-min time point was conducted. At the 60 min time point, the two-way ANOVA revealed main effects of Treatment [F(1,24) = 20.5, p < 0.001] and Virus [F(1,24) = 6.5, p = 0.017], as well as a significant Treatment × Virus interaction [F(1, 24) = 8.9, p = 0.006] confirming significantly lower body temperature in the CNO-injected vGlut2-Cre mice transfected with the DIO-hM3Dq DREADD versus other groups.

Fig. 4. Activation of the EWcp decreases body temperature in both male and female mice.

Fig. 4.

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Following recovery from stereotaxic surgery, Vglut2-Cre mice that had received either DIO-hM3Dq of eGFP control viruses into the EWcp were treated with CNO or vehicle. Body temperatures were recorded 30, 45, and 60 minutes post i.p. injection. A) Experimental timeline. B) CNO-mediated activation of Vglut2-expressing neurons in hM3Dq-transfected mice resulted in a robust decrease in body temperature over time.

4. Discussion

Our studies demonstrate that acute chemogenetic modulation of neurons within the EWcp leads to significant decreases of ETOH drinking in the 2-BC paradigm. Specifically, activation, but not inhibition of the EWcp leads to a significant decrease in ETOH intake. Furthermore, our studies provide evidence that the subpopulation of Vglut2-expressing EWcp neurons acts to inhibit ETOH intake and to decrease body temperature.

Numerous studies using various approaches have previously identified the EWcp, and specifically Ucn1 neurons in the EWcp, as being highly sensitive to ETOH and involved in regulation of ETOH self-administration (Bachtell et al., 2004; Giardino et al., 2011, 2017). Our finding that chemogenetic activation, but not inhibition, of the EWcp leads to decreased ETOH intake expands this evidence. Surprisingly, however, immunohistochemical analyses of the EWcp from the first of our experiment found no mCherry, and thus no DREADD expression, in Ucn1-positive cells. The lack of DREADD expression in Ucn1 neurons in the EWcp suggests that the AAV8 serotype we used lacks the ability to transfect Ucn1-positive neurons, at least with the titer and volume that was used. One possible explanation for the lack of AAV8 transfection in Ucn1-expressing neurons is that these neurons in the EWcp do not express the receptors required for AAV transfection. Importantly, our pilot experiments also showed lack of transfection of Ucn1 neurons following injections of AAV2, AAV5 and AAVDJ viral serotypes (data not shown). To date, the exact molecular mechanisms involved in AAV transfection of cells have not yet been deciphered. The laminin receptor has been shown to be a potential receptor for AAV8 as well as AAV2, 3 and 9 (Akache et al., 2006). In addition, recent studies pointed to a universal receptor, KIAA0319 (or AAVR), as essential for cell binding for multiple AAV serotypes. (Pillay et al., 2016). Currently, it is not known if Ucn1 neurons in the EWcp express AAV receptors, and future work investigating this possibility is required. Interestingly, a prior RNA viral interference experiment showed that Ucn1 neurons can be transfected with lentiviruses (Giardino et al., 2017). Future studies could use lentiviral vectors to chemogenetically manipulate the activity of EWcp Ucn1 neurons, in order to further assess their involvement in ETOH intake, particularly in relation to Vglut2-expressing neurons. Future work using a viral vector with a CamKII promoter may also provide an alternative approach to target neurons in the EWcp, particularly as excitatory glutamatergic neurons have been shown to express CamKII (Basu et al., 2008; Jones et al., 1994).

Our observation that Ucn1-positive neurons did not express DREADDs, combined with the significant decrease in ETOH intake observed in these mice indicated that a different population of EWcp neurons must be involved in the inhibition of ETOH intake. This idea was confirmed in a subsequent experiment where chemogenetic activation of the EWcp decreased ETOH intake, in both Ucn1 KO and WT littermates. During the 12-day period in which mice had 24hr access to increasing concentration of EtOH we observed no differences between Ucn1 KO and WT mice. This finding is not entirely surprising, as previous work in our lab has shown that neither genetic nor shRNA deletion of Ucn1 alters 3–10% ETOH intake when mice have 24hr access to ETOH (Giardino et al., 2017). Although the difference was not significant, we also found that Ucn1 KO mice drank slightly less than their WT littermates during the 4hrs post CNO injection. This difference in intake is similar to what has been previously reported. Indeed, Giardino and colleagues found that Ucn1 KO and WT mice differ in their 20% EtOH intake primarily during the initial hours of the dark cycle (Giardino et al., 2017). Here, the 4hr-intake period reported in Fig. 2C occurred during the first 4hrs of the dark cycle. Future work in which the EWcp is manipulated at various time points through the dark cycle, between these two genotypes, may help determine if circadian rhythms affect chemogenetic-mediated decreases in intake.

Our findings also indicate that EWcp Vglut2-expressing neurons are involved in the regulation of ETOH intake. Vglut2 is a well-known marker of glutamatergic neurons (Vigneault et al., 2015). The involvement of the glutamate system, and/or changes in glutamate transmission during and after ethanol intake have been extensively reported in various regions that receive input from the EWcp, including the CeA (Roberto et al., 2004; Zhu et al., 2007), lateral hypothalamus (Chen et al., 2013; Wei et al., 2015) and the bed nucleus of stria terminalis (Kash et al., 2009; Wills et al., 2012). Given our findings, it is thus possible that increased activity in the glutamatergic projection from the EWcp to one or several of these regions decreases ETOH intake. Previous work in our lab has shown that the majority of ETOH-activated neurons in the EWcp are Ucn1-positive (Anacker et al., 2014; Bachtell et al., 2002a; Ryabinin et al., 2003), indicating that ETOH may increase neuronal activity in Ucn1-expressing neurons. One possibility that should be considered is that the decrease in intake observed after activation of Vglut2-expressing neurons could be mediated by the interaction between these neurons and those that express Ucn1 in the EWcp. Understanding how these two populations of neurons interact may provide insight into how ETOH intake is mediated by the EWcp. Ultrastructural work has shown that both D- and L-type asymmetrical synapses make contact with Ucn1 neurons in the EWcp (Van Wijk et al., 2009), and as asymmetrical synapses are believed to be excitatory (DeFelipe & Fariñas, 1992), one possibility is that these glutamatergic neurons may act to regulate Ucn1-expressing neurons. Furthermore, Van Wijk and colleagues reported that the sizes of vesicles found on these asymmetrical contacts were consistent with those that have been shown contain glutamate (Van Wijk et al., 2009). Although Ucn1-neurons were not transfected by the AAV carrying the hM3Dq receptor in Experiment 1, it is possible that some of the observed effect on ETOH intake occurs via interactions between transfected neurons and Ucn1-expressing neurons. Future electrophysiological studies investigating how neuronal activity in Ucn1-expressing neurons is altered following activation or inhibition of these glutamatergic neurons will undoubtedly provide much needed additional information.

Vglut2-expressing neurons in the EWcp have not been previously studied in relation to actions of addictive substances. A recent study however, found that Vglut2 neurons in the vicinity of the EWcp, also known as the perioculomotor area (pIII), are important for the regulation of non-rapid eye movement (NREM) sleep (Zhang et al., 2019). Zhang and colleagues found that Vglut2 neurons of pIII form at least two subpopulations of neurons, containing either CCK or calcitonin gene-related peptide alpha (CALCA) (Zhang et al., 2019). It is possible that some of the Vglut2 neurons studied here and those studied by Zhang and colleagues may be the same populations of neurons. Importantly however, the decrease in intake following activation of Vglut2 neurons in our study could not be due to potential sleep-promoting effects of these neurons as the decrease in intake was selective for ETOH, and was not observed for water or sucrose intake. Interestingly, we found that activation of Vglut2-expressing neurons only decreased intake during the first 2hrs after CNO treatment. This is in contrast to our first two experiments using the non-specific hM3Dq DREADD, in which we saw decreased intake up to 4hrs after CNO injections. The difference in the time-course of effects of CNO in experiments 1 and 2, compared to experiment 3, suggests that other EWcp neuronal populations that do no express Vglut2, and thus were only transfected in experiments 1 and 2, may also be involved in the regulation of ETOH intake. Several additional neuropeptides and neurotransmitters are expressed in varying subpopulations within the EWcp, and may contribute to the regulation of EtOH intake. These include CCK (Innis & Aghajanian, 1986; Zhang et al., 2019), substance P (Maciewicz et al., 1983), the stress-related peptide PACAP (Gaszner et al., 2012), and dopamine (Bachtell et al., 2002b). Future work further characterizing the involvement of these specific subpopulations of EWcp neurons in ETOH intake is still required.

In addition to playing a critical role in the regulation of ETOH intake, a number of previous studies have also implicated the EWcp as being involved in thermoregulation. Initial reports provided evidence that the EWcp is sensitive to temperature changes, as mice exposed to either cold or warm ambient temperatures showed an increase in c-Fos expression in the EWcp (Bachtell et al., 2003). Furthermore, electrolytic lesions of the EWcp were shown to blunt the ETOH-induced hypothermic response seen in mice following a single injection of ETOH, suggesting that the EWcp may also regulate body temperature (Bachtell et al., 2004). More recently, pharmacological inhibition of the EWcp was shown to increase body temperature (Zuniga et al., 2020), and although pharmacological inhibition of the EWcp did not specifically target glutamatergic neurons, it is possible that this recent finding was driven, at least partially, by the inhibition of Vglut2-positive neurons. Thus, when considered in light of these previous studies, our finding that activation of Vglut2-positive neurons in the EWcp decreases body temperature suggests that the EWcp may be able to regulate body temperature bidirectionally. Interestingly, several lines of evidence have shown that Ucn1 within the EWcp (Bachtell et al., 2002a), and its downstream targets (Turek & Ryabinin, 2005) plays an important role in thermoregulation. Future work in which the EWcp and Vglut2 neurons specifically are inhibited will help determine if the same populations of neurons within the EWcp can indeed regulate temperature bidirectionally. Additionally, future studies investigating the interactions between Ucn1- and Vglut2-positive neurons in the EWcp may help elucidate the roles that these two distinct neuronal populations may play in regulating body temperature. Whether the effects of EWcp activation on ETOH intake and body temperature are dependent or independent of each other should be also addressed in future studies, as it is possible that changes in body temperature may alter ETOH intake.

In summary, the studies presented here demonstrate that chemogenetic activation of non-Ucn1-expressing neurons in the EWcp decreases ETOH intake. Specifically, Vglut2-expressing neurons were found to mediate this effect, as specific activation of these neurons decreased ETOH intake while not affecting water or sucrose consumption. Additionally, our studies suggest that Vglut-2 expressing neurons in the EWcp may regulate body temperature. Together, our findings suggest that the EWcp may be a promising future therapeutic target for the treatment of alcohol use disorders. Future studies should focus on disentangling the roles of specific subpopulations within the EWcp and their projections in the regulation of ETOH-related behaviors.

Supplementary Material

1
2

Highlights.

  1. Activation, but not inhibition, of the EWcp using DREADDs decreased alcohol intake in mice.

  2. AAVs did not lead to DREADD expression in Ucn1-containing neurons of the EWcp.

  3. Activation of the EWcp using DREADDs decreased alcohol intake in Ucn1 knockout mice.

  4. Activation of Vglut2 neurons of the EWcp using DREADDs inhibited alcohol intake.

  5. Activation of Vglut2 neurons of the EWcp using DREADDs reduced body temperature.

Funding and Acknowledgements.

AZ was supported by NIH NIAAA T32 training grant AA007468 and a Diversity Supplement awarded to NIH NIAAA R01 AA07702. MLS was supported by an NIH NIAAA F31AA022824 award. MC and AER were supported by NIH NIAAA R01 AA019793 and R01 AA025024.

Footnotes

The authors declare no conflicts interest.

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