Neurokinin-1 receptors in the nucleus accumbens shell influence sensitivity to social defeat stress and stress-induced alcohol consumption in male mice

Abstract

Chronic social defeat stress (SDS) is a widely employed preclinical model of depression involving repeated exposure to physical defeats using a resident-intruder model in male mice. Exposure to SDS induces depressive-like phenotypes including anhedonia, social withdrawal, and increased drug and alcohol consumption. Previously, we found that expression of the neurokinin-1 receptor (NK1R) is increased in the nucleus accumbens (NAC) of mice that are sensitive to this stressor and increase their alcohol intake. The NK1R is the endogenous receptor for the neuropeptide substance P (SP) and plays a prominent role in stress, anxiety, and addiction. In the present study, we assessed changes in NK1R protein levels in the NAC shell and implemented viral vector strategies to demonstrate a functional role of the NK1R in sensitivity to SDS. Specifically, we found that NK1R protein levels were increased in the NAC shell following SDS exposure. Next, we found that NK1R overexpression in the NAC shell increased the sensitivity to SDS and stress-induced alcohol consumption. Together, these experiments provide evidence for a role of the NK1R in the NAC shell in the sensitivity to SDS and the subsequent escalation in alcohol intake.

1. Introduction

Alcohol Use Disorder (AUD) is often co-expressed with other psychiatric conditions such as anxiety and depression (1, 2). Specifically, among individuals with AUD, the odds ratio for lifetime comorbid depression is almost 4, indicating that depression is significantly more common in individuals with AUD compared to the general population (3, 4). This relationship is bidirectional, as approximately 25% of AUD patients present with comorbid depression (5). Not only does AUD increase the risk for, and severity of, depression, but a history of depression conversely increases the risk of subsequent AUD (6). Thus, the study of mechanisms that mediate this comorbid relationship between alcohol abuse and depression has the potential to help improve treatment outcomes for patients that co-express these conditions.

Over the last several years, our group and others have shown a role of the neurokinin-1 receptor (NK1R) in alcohol seeking behavior. The NK1R is the endogenous receptor that binds most strongly to the neuropeptide substance P (SP; 7). SP and the NK1R have an integral role in pain processing, opiate reward and reinforcement, stress, anxiety, and alcohol seeking (8–12). Specifically, we have shown that the NK1R mediates stress-induced relapse-like behavior and escalated alcohol consumption in rodent models (13–19). This effect on stress-induced relapse-like behavior extends to other drugs of abuse, including cocaine and oxycodone (17, 20). In general, NK1R antagonism does not affect baseline alcohol consumption at doses that lack off target effects, but it does suppress alcohol intake that is potentiated by conditions such as stress exposure, genetic selection, and intermittent access schedules (16, 19, 21, 22). These effects are primarily mediated by NK1R activation in the amygdala and striatum (13–16, 19, 23). In line with these findings, a recent study showed increased ability of NK1R to stimulate GABA neuron activity in the central nucleus of the amygdala following chronic alcohol exposure (24). These preclinical findings are supported by human studies which have reported efficacy of NK1R antagonists on alcohol craving, as well as genetic associations of the TACR1 gene (gene for NK1R) with alcohol dependence and sensitivity to alcohol paired cues (25–27).

Related to depression, there are extensive preclinical data demonstrating a role of the NK1R in depressive-like behavior and anxiety (11, 12, 28). Behavioral data in these domains are bolstered by a considerable body of work showing that SP and the NK1R can mediate monoamine signaling and neuronal activity in critical, stress-related brain regions (28–36). NK1R antagonists held great promise as a new generation of antidepressant pharmacotherapy following positive results in clinical trials in the mid-1990s (37). However, following trials with ambiguous or negative results, most clinical research into the NK1R as an antidepressant treatment was abandoned (38, 39). More recent research has shown renewed promise and has demonstrated that antidepressant effects of NK1R antagonists require high levels of receptor occupancy, which can be achieved using sufficient doses of newly developed compounds targeting this receptor (40–42).

A major preclinical rodent model used to study depression in the laboratory is social defeat stress (SDS; 43, 44, 45). SDS exposure induces anxiety-like behaviors in a variety of testing models, and depressive-like behaviors including anhedonia and social avoidance (46). Pro-depressive phenotypes of SDS-exposed mice are sensitive to chronic, but not acute, treatment with standard antidepressant drugs (43, 45). While there is some discrepancy in the literature, SDS exposure generally increases drug and alcohol intake in mice that are sensitive to this stressor. For example, chronic SDS increases alcohol consumption (47–54), conditioned place preference (55), self-administration (56), and motivation to seek alcohol (56). Some studies report no change or decrease after defeat stress (51, 57–60), but overall most studies report an increase in alcohol consumption after SDS. We have shown this effect in a recent study, and also observed that increased levels of the transcript for the NK1R in the ventral striatum of mice that show reduced social exploration following SDS exposure (23). The ventral striatum includes the nucleus accumbens (NAC), which is known to mediate stress and drug seeking. Other groups have demonstrated a clear role of the NAC in the behavioral and molecular response to SDS (61–63), and we have found that activation of this region facilitates stress-elicited alcohol seeking and alcohol reward (15, 64). For example, findings from our group have shown that the NK1R in the NAC shell subregion promotes stress-induced alcohol seeking in the reinstatement model (15), and thus we chose to focus on this specific area in the experiments presented here. Taken together, the literature suggests that the NK1R in the NAC may mediate responses to SDS and potentially its ability to induce escalated alcohol consumption.

In the current study, we extend our prior findings to examine the functional role and subregional focus of NK1R actions in the NAC in SDS-induced behaviors. We assessed both behavioral sensitivity to SDS as well as SDS-induced escalation in alcohol intake using viral vector-based overexpression methods. Our results suggest that NK1R activity in the NAC shell contributes to SDS-induced depressive-like behavior and escalated alcohol intake following stress exposure in male mice.

2. Methods

2.1. Animals

Male C57BL6/J mice (8–10 weeks of age, Jackson Laboratory, Bar Harbor, ME) were used as experimental subjects. Retired breeder male CD-1 mice (4–5 months of age, Charles River, Wilmington, MA) were used as SDS aggressors. Due to sex-specific differences in territorial aggression and the SDS protocol being replicated and highly validated using male C57BL6/J mice, only male subjects were used in this study. Mice were allowed at least 1 week of habituation to the University of Georgia College of Veterinary Medicine vivarium before experiments began. Mice were housed on a 12hr light cycle (on at 1:00 and off at 13:00). Food and water were available ad libitum. All procedures were in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Georgia.

2.2. SDS

SDS was performed as previously described (54) and was based on the protocol described by Golden and colleagues (44). Prior to SDS, male CD-1 mice were screened for aggressive behavior by placing a screener C57BL/6J mouse (not used in the study) into the home cage of the CD-1 mouse for 3-min for 4 consecutive days. Aggressors were selected based on the following criteria: the CD-1 mouse must initiate an attack in at least 2 consecutive sessions and the latency to first display of aggressive behavior must be less than 60 seconds. Aggressors that passed aggression criteria were placed into a large cage (26.7cm (w) × 48.3cm (d) × 15.2cm (h); product number: N40, Ancare, Bellmore, NY) with a clear, perforated divider 72 hours prior to the start of SDS. Defeat sessions consisted of placing C57BL6/J mice into the homecage of the CD-1 mouse for five minutes, after which the defeated mice were placed on the opposite side of the partition for the remainder of the 24 hours. This non-physical defeat phase allows for olfactory, visual, and auditory cues to be exchanged between the C57BL6/J mouse and the aggressive CD-1 it just encountered. This process then repeats for 10 consecutive days with the C57BL6/J mice encountering a novel aggressor each day. Control mice were housed in pairs in identical hamster cages with a mouse on either side of the perforated divider. Mice were rotated each day to mimic the novel housing conditions experienced by the SDS mice but did not get physically defeated nor encountered any CD-1 mice throughout the 10 days. Defeats occurred at the end of the light cycle (as close to the beginning of the dark cycle as possible) in order to visualize any injuries. If blood was visible, the mice were separated. All mice had ad lib access to food and water except during the 5 minute aggressive encounters.

2.3. Social Interaction (SI) test

Immediately following the final defeat session, mice were singly housed in regular mouse cages with food and water available ad libitum. Approximately 24 hours following the last defeat exposure (at the beginning of the dark cycle), mice underwent the SI test, as previously described (23, 65). This test consisted of 2 150-second trials separated by 30 seconds, the first without a novel CD-1 target mouse present, and the second with a target mouse present in an enclosure in the predetermined interaction zone. Time spent in the interaction zone during each trial was recorded, and time in the interaction zone with the target mouse present was divided by the time when the target was absent to obtain the SI ratio. SI tests were scored by an observer blind to treatment.

2.4. Subthreshold SDS

Mice were exposed to 3 defeat sessions of 5-minute duration, separated by 15 minutes each. During each defeat session, mice were placed into the homecage of a novel male CD-1 mouse previously screened for aggressive behavior. Between defeat sessions, mice were returned to their homecage to recover until the next defeat session started. This procedure has been utilized in previous studies from our lab (65).

2.5. Two Bottle Choice (2BC)

Mice were presented with two bottles in their homecage, one containing water and one containing a 20% ethanol solution on a continuous access schedule, according to previously utilized methods in our lab(23). Access began at 20% alcohol with no ramping of alcohol concentration or sucrose/saccharin fade. 95% ethanol (Decon Labs, Inc., King of Prussia, PA) was diluted to a 20% v/v solution in tap water. Bottles were weighed at the same time each day, and g/kg consumption was calculated based on each mouse’s body weight. Duration and timing of drinking is included in experimental timeline figures.

2.6. Immunofluorescence

Mice were perfused with 4% paraformaldehyde 24 hours after the final SDS exposure. Brains were sectioned to 30μm thickness using a cryostat (Leica) and stored in cryoprotectant at −20°C. Sections were floated in net wells and washed 3 times in tris-buffered saline (TBS), 5-min per wash. Sections were permeabilized for 30-min in TBS with 0.3% Triton-X (TBS-Tx). Following permeabilization, tissue sections were blocked using 5% Normal Donkey Serum in TBS (TBS-NDS) and washed 3 times in TBS. Sections were incubated overnight at 4° C with NK1R rabbit antibody (1:1000; Millipore, AB5060) in TBS-NDS, and then sections were washed 3×5-min in TBS. Tissue sections were incubated at room temperature in fluorescent (633nm) 2°antibody (goat anti-rabbit; Invitrogen cat# A21070) for 2-hr then washed 3 times with TBS. Tissue was mounted on slides and coverslipped with Vectashield. Using a Nikon A1 confocal microscope, a z-stack image of 10μm of tissue (11 steps @ 1μm each) was collected for the NAC shell. Confocal imaging settings were kept constant for all samples. Using ImageJ, image stacks were summed together, the background subtracted, and then the fluorescence intensity determined.

2.7. Virus Infusion Surgery

AAV1-NK1R-GFP was produced by Brandon Harvey and Christopher Ritchie at the National Institute on Drug Abuse using PCR amplification of the NK1R (TACR1) cDNA from a lentiviral vector containing rat TACR1 (GeneCopoeia; # CS-Rn10116-Lv156–01) and subsequent insertion upstream of the IRES-EGFP element within pAAV CMV-IE MCS IRES EGFP (Addgene #102936). The resulting construct pAAV CMV-IE NKR1 IRES GFP (Addgene #102935) was packaged as an AAV serotype 1 and purified by affinity chromatography as previously described (66). The resulting vector is referred to as “AAV1-NK1R-GFP”. AAV1-NK1R-GFP (viral titer: 3.5 × 10¹² vg/ml) or control AAV1-GFP (viral titer: 5.3 × 10¹² vg/ml) virus was bilaterally infused into the NAC shell of C57BL6/J mice using a stereotaxic instrument (Stoelting, Wood Dale, IL). Microinfusions were given through a blunt end Hamilton Neuros Syringe (Reno, NV) mounted to the stereotax and controlled by a Precision syringe pump (World Precision Instruments, Sarasota, FL). Infusions were given over 5 minutes and syringes were left in place for 5 minutes following infusion to prevent aspiration along the needle tract. For the effect of NK1R overexpression on SDS sensitivity, infusions were 0.5 μl in volume and targeted to the coordinates AP: +1.7mm, ML: ±2.3mm, DV: −4.7mm (20° angle). For the effect of NK1R overexpression on alcohol consumption, infusions were 0.25 μl in volume and targeted to the coordinates AP: +1.6, ML: +/− 0.7, DV: −4.4 (0° angle). While the coordinates and angle were slightly different between these experiments, in both cases infusions were targeted to the medioventral portion of the medial NAC shell, with some spread into the NAC core. After 4 weeks of recovery to allow for full viral vector expression, mice were exposed to subthreshold SDS and then assessed for SI or allowed access to alcohol. After the completion of experiments, mice were sacrificed, and placements of viral infusion were checked via fluorescent microscopy. If the infusion in both hemispheres was placed outside of the NAC shell, the animal was removed from the study. This virus and specific methods have been used in our previous work (13). This manuscript also includes images of receptor upregulation following viral infection.

2.8. Statistics

Statistical analyses were preformed using GraphPad Prism software. Independent samples t-test or two-way ANOVA were used for analysis as stated in the Results section. Bonferroni posthoc tests were performed when appropriate.

3. Results

As stated above, we have previously observed increased expression of the NK1R in homogenates from the ventral striatum using qPCR methods. To assess the effect of SDS on NK1R protein levels in the NAC shell specifically, mice were exposed to SDS (n=13) or left unstressed (N=8), and were then sacrificed 24 hours after the final session. NK1R in NAC shell sections were labeled with immunofluorescence. An unpaired t-test revealed an increase in NK1R protein in the NAC shell compared to controls following SDS exposure (t₁₉=2.4, p=0.02; Fig 1A–C). These results indicate that general expression changes in the ventral striatum are associated with increased receptor protein levels in the NAC shell subregion.

Figure 1. SDS exposure induces increased NK1R expression in the NAC shell.

(A) Time line of experiment. (B) Increased expression of NK1R was observed in the NAC shell, as measured by fluorescent density. Tissue was extracted following the final defeat session. *p<0.05. (C) Representative staining for the NK1R in the NAC shell in SDS exposed mice and unstressed controls.

We next investigated the effects of NK1R overexpression in the NAC shell on sensitivity to SDS using the subthreshold SDS protocol (44, 54). This one-day stress exposure does not typically induce depressive-like behavior in mice, and allows for the investigation of prodepressant manipulations on stress sensitivity (44). C57BL6/J mice were stereotaxically injected with either AAV1-NK1R-GFP (n=7) or AAV1-GFP (n=7) in the NAC shell and were allowed to recover for 4 weeks. Following this recovery period, mice were exposed to subthreshold SDS and tested for SI 24 hours later. Mice infused with the NK1R overexpression virus in the NAC shell displayed an increase in stress sensitivity, as evidenced by a decrease in SI following subthreshold SDS compared to mice infused with the GFP control virus (t₁₂=2.5, p=0.03, Figure 2A–C). Importantly, SI behavior was unchanged in a separate cohort of mice that was infused with AAV1-NK1R-GFP (n=5) as compared to AAV1-GFP infused mice (n=5) in the absence of stress (mean ±SEM of SI ratio: AAV1-GFP = 1.18 ± 0.13, AAV1-GFP-NK1R = 1.03 ± 0.19; t₈=0.67, p=0.52).

Figure 2. Overexpression of the NK1R in the NAC shell increases sensitivity to subthreshold SDS.

(A) Timeline of experiment (B) Mice infused with the NK1R overexpression virus in the NAC shell displayed an increase in stress sensitivity as evidenced by a decrease in social interaction (SI) following subthreshold SDS compared to mice infused with the GFP control virus (p=0.03). (C) Representative image of virus expression. Dotted line indicates approximate border of NAC core/shell. Mouse brain schematic obtained from Allen Brain Atlas (https://mouse.brain-map.org/). *p<0.05

Because SDS increases alcohol intake and NAC shell NK1R expression, we hypothesized that NK1R upregulation in this region, even in the absence of SDS, would drive increased alcohol intake. Thus, we infused AAV1-GFP-NK1R (n=9) or AAV1-GFP (n=10) virus in the NAC shell and measured subsequent alcohol intake. Two-way ANOVA with the factors of day (within subjects) and virus treatment (between subjects) revealed no difference in alcohol intake between mice with NK1R overexpression and those injected with the control virus (main effect of virus F(1,17)=0.32, p=0.58; interaction effect F(26,442)=0.80, p=0.7499; Figure 3A–B). Two way ANOVA of alcohol preference did not detect any significant effects (main effect of virus F(1,17)=0.08, p=0.78; interaction effect F(26,441)=0.81, p=0.73; Figure 3C). Representative image of viral infusion shown in Figure 3D.

Figure 3. Overexpression of the NK1R in the NAC shell does not affect alcohol intake in the absence of stress.

(A) Time line of experiment. Alcohol consumption was tracked over 4 weeks. Viral overexpression of NK1R in NAC shell did not affect voluntary alcohol intake (B) or preference (C) in the absence of stress. (D) Representative image of virus expression. Dotted line indicates approximate border of NAC core/shell.

We then exposed these mice to a subthreshold defeat and found that this treatment induced increased alcohol intake in NK1R overexpressing mice. Specifically, two-way repeated-measures ANOVA indicated a main effect of day (F(9,153)=4.4, p<0.001) and a virus x day interaction (F(9,153)=2.1, p=0.03; Fig 4A). The main effect of virus (F(1,17)=2.18, p=0.16) did not reach statistical significance. Post-hoc Bonferroni’s tests did not identify significant differences between treatment groups on any individual day. Analysis of alcohol preference did not reveal significant main effects (main effect of day F(9,81)=1.8, p=0.08; main effect of virus F(1,9)=0.76, p=0.41) or interaction (F(9,69)=1.6, p=0.13; Figure 4B). However, we observed increased alcohol consumption after stress when compared to before stress specifically in the NK1R overexpressing group. An average of the last 3 days prior to stress was used to calculate pre-stress drinking and average of the first 3 days after stress was used for post-stress drinking (Figure 4C). Two way ANOVA revealed a main effect of stress (F(1,17)=7.56, p=0.01) and a nearly significant main effect of virus treatment (F(1,17)=4.0, p=0.06), and trend level stress x virus interaction effect (F(1,17)=3.20, p=0.09). Post hoc Bonferroni test indicated a significant difference between pre-stress and post-stress drinking for NK1R overexpressing mice (p=0.01), but not GFP control expressing mice (p=0.99). There was also a significant difference between viral treatments after stress (p=0.02), but not prior to stress (p=0.95). Analysis of alcohol preference using this design did not detect any main effects (main effect of stress F(1,17)=0.22, p=0.65; main effect of virus F(1,17)=1.2, p=0.28) or interaction (F(1,17)=2.4. p=0.14; Figure 4D). Taken together, this suggests that NK1R upregulation in the NAC shell increases stress-induced alcohol intake, at least transiently.

Figure 4. Overexpression of the NK1R in the NAC shell increases alcohol intake following subthreshold SDS.

Daily consumption of alcohol (A) and preference (B) after subthreshold SDS exposure. Average of alcohol consumed (C) and preference (D) was taken for the 3 days prior to stress and the 3 days following stress, and compared using two way ANOVA. *p<0.05 compared to NK1R OE pre-stress, #p<0.05 compared to GFP post-stress.

4. Discussion

In these experiments, we identify a role of the NK1R in the NAC shell as a mediator of SDS-induced depressive-like behaviors. Specifically, NK1R expression is upregulated in this region following exposure to SDS, which advances our prior work that demonstrated increased levels of the transcript for the NK1R in the ventral striatum generally. This result informed the site-directed viral overexpression experiments, and supported our hypothesis that the NAC shell is a critical node in the NK1R effect on stress and depression-like behavior. Congruent with this, overexpression of the NK1R in the NAC shell using a viral vector increases social avoidance following subthreshold SDS, indicating increased susceptibility to this stressor. We found that viral-driven overexpression of the NK1R did not alter SI behavior or increase alcohol intake in the absence of stress, but did enhance alcohol drinking that followed a brief re-exposure to defeat. Taken together, these results suggest that the NK1R in the NAC shell mediates the longterm effects of SDS on social behavior, and that this receptor contributes to acute escalations in alcohol intake following stress exposure.

As outlined in the introduction, there is an extensive literature showing a role of the NK1R in depression-related behavior. Our findings are consistent with this body of work. We observed an effect of the NK1R that appears to be localized in part to the NAC shell, which is somewhat novel in regards to previously reported NK1R effects in depression models. Earlier studies have suggested a role of the NK1R in the lateral septum, raphe nuclei, and lateral habenula, among other regions (28, 34, 67); the role of NK1R in the NAC in depression-related behaviors has not been specifically assessed. However, the importance of the NAC is supported by much prior work on the SDS model specifically (see below). Furthermore, prior work has demonstrated a role of the NK1R in NAC excitability(68). One consideration is that we utilized an AAV1 vector for delivery of our NK1R overexpressing virus. It has recently been reported that this serotype of AAV has anterograde transsynaptic properties in neurons (69). Thus, it is possible that we drove NK1R upregulation in regions to which the NAC projects, in addition to the NAC itself. We note, however, that the NK1R has been found to mediate depression-like behavior in multiple regions of the forebrain (see above). Future experiments with other viral serotypes will examine the anatomical specificity of NK1R upregulation on alcohol consumption and depression-like behavior. While we demonstrate a role of the NAC shell subregion in this behavior, we did not examine the impact of the NAC core, the other major subdivision of the NAC, and it is quite possible that NK1Rs in this region have a similar effect on SDS-induced phenotypes. Overall, it appears that the NK1R is recruited in multiple brain regions to mediate the behavioral phenotypes of depressive-like behavior, especially those that are induced by SDS.

One important point to note is that our work only used male mice, as it is difficult to use traditional resident-intruder-based stress designs in female C57BL6/J mice due to a lack of territorial aggression. Future work will attempt to address this shortcoming, which is of utmost importance given the higher incidence of depression observed in women clinically. A few groups have begun to address this issue and have found interesting results that generally coincide with mechanisms observed in male mice (70–73). It is unclear if parallel effects will be observed in female mice in regards to the role of NK1R in these behaviors, as the vast majority of prior preclinical work on NK1R in depression-like behavior and alcohol consumption has used only male rodents. However, our recent work has demonstrated reduced social interaction and increased alcohol consumption in female mice following vicarious defeat stress exposure (74). We found that escalated alcohol consumption was reduced by NK1R antagonism, but this treatment was effective in non-stressed female mice as well. Thus, it is possible that NK1R upregulation using our viral vector would be capable of inducing escalated consumption in female mice in the absence of stress, and this will be tested in future studies.

Two recurring elements of the SDS literature include the role of the NAC and the importance of neuroinflammatory mediators such as cytokines (62, 75–81). For example, induction of the inflammation-related transcription factor, nuclear factor kappa B (NFkB) in the NAC is a mediator of SDS-induced alterations in behavior, physiology, and dendritic spine remodeling (61, 63). As outlined above, our data show a role of the NK1R in the NAC shell in our model. Linking this to inflammatory responses, we have previously reported that NK1R antagonism can attenuate cytokine expression and depressive-like behavior following injection of the immune stimulator lipopolysaccharide (82). This suggests that the NK1R may serve as a link between stress and expression of inflammatory mediators, and that this may occur in the NAC as well as other brain regions.

In these experiments, our initial hypothesis was that overexpression of the NK1R would phenocopy SDS exposed mice and increase alcohol consumption in the absence of stress. However, we did not see any change in alcohol consumption in virus infused, stress-naïve mice. However, increased alcohol intake was observed in NK1R overexpressing mice following exposure to a brief SDS challenge. This escalated intake was sustained for a few days, but not maintained long term. It is possible that mild stress induces a temporary NK1R upregulation that subsides over time. It is hypothesized that chronic stress would induce a longer lasting NK1R upregulation and escalated alcohol intake, as seen after 10 SDS exposure(23). The induction of escalated alcohol consumption in stress-naïve mice may require NK1R upregulation in additional structures. Of note, we have previously reported increased operant self-administration of alcohol in Wistar rats following viral-driven overexpression in the central nucleus of the amygdala (13). It is also important to note that our virus will be taken up and express the NK1R in many cell types. We did not assess the cell type in which NK1R is upregulated in our post-SDS assessments, but it is known that the NK1R is found in GABAergic and cholinergic cell bodies, as well as presynaptic terminals in the NAC (68, 83–86). Discrepancies in the behavioral effects of virus driven upregulation and SDS driven upregulation may result from such differences. Additionally, future experiments will pharmacologically target NK1R signaling at the time of alcohol consumption after SDS, to determine if NK1R inhibition at this point can attenuate alcohol intake, reversing SDS-induced escalation.

In conclusion, these results support a functional role of the NK1R in the NAC shell in the expression of SDS-induced depressive-like phenotypes and escalated alcohol intake. This suggests that NK1R-targeted treatments may be potential candidates for the development of novel antidepressants, and supports continued exploration of these agents in drug development.