Voluntary binge- patterned alcohol drinking and sex- specific influences on monoamine- related neurochemical signatures in the mouse gut and brain

Background: Altered monoamine (i.e., serotonin, dopamine, and norepinephrine) ac tivity following episodes of alcohol abuse plays key roles not only in the motivation to ingest ethanol, but also physiological dysfunction related to its misuse. Although monoamine activity is essential for physiological processes that require coordinated communication across the gut– brain axis (GBA), relatively little is known about how alcohol misuse may affect monoamine levels across the GBA. Therefore, we evalu ated monoamine activity across the mouse gut and brain following episodes of binge-patterned ethanol drinking. Methods: Monoamine and select metabolite neurochemical concentrations were analyzed by ultra- high- performance liquid chromatography in gut and brain regions of female and male C57BL/6J mice following “Drinking in the Dark” (DID), a binge-patterned ethanol ingestion paradigm. Results: First, we found that alcohol access

neurochemical producing and utilizing GI microbes, thereby altering monoamine levels in host intestinal tissues (Leclercq et al., 2014;Szabo & Saha, 2015). The gut produces nearly 50% of the body's dopamine and 90% of the body's serotonin (Bellono et al., 2017;Eisenhofer et al., 1997); thus, altered monoamine activity following episodes of alcohol abuse could not only have an impact on intestinal function and communication across the entire GBA but also contribute to GI dysfunction. For example, alcohol ingestion can exacerbate symptoms of GI disorders characterized by abnormal monoaminergic activity (e.g., Crohn's disease, ulcerative colitis, and irritable bowel syndrome) (Mittal et al., 2017). Moreover, disturbances to the balance of monoamines in the gut can influence cognitive processes that require monoaminergic signaling (Borghammer, 2018;Breit et al., 2018;Davey et al., 2013;McVey Neufeld et al., 2019;Suarez et al., 2018). Evidence also suggests intestinal nerves may be initial target of selective serotonin reuptake inhibitors (i.e., SSRIs) capacity to mitigate rodent depression-like behavior mediated by abnormal monoaminergic activity in the brain (Desbonnet et al., 2015;Liang et al., 2015;McVey Neufeld et al., 2019). Therefore, alcohol-induced changes to gut monoamine concentrations may modify communication across the entire GBA network, potentially increasing the risk for the development of neurobiological disorders (Bonaz et al., 2017;Kelly et al., 2016;Lionnet et al., 2018). However, despite the critical roles of monoamines in GBA function and dysfunction, monoaminergic changes in the gut remain uncharacterized following alcohol abuse.
Ingesting high volumes of alcohol can also potently alter the activity of monoamines within several brain pathways including reward, motor, the hypothalamic-pituitary-adrenal axis (HPA), and limbic system. In fact, the altered activity of monoamine neurotransmitters in these circuits is linked to the motivation to misuse alcohol, risk for the development of neurobiological disease, and alcohol-related cognitive dysfunction (Koob & Volkow, 2016).
Research into the impact of alcohol abuse on brain monoamines has predominately focused on dopaminergic activity in reward circuit structures, such as the ventral tegmental area and the nucleus accumbens (Koob, 2014;Lovinger & Alvarez, 2017;Weiss et al., 1996).
Monoamine activity in other brain regions that comprise parts of the limbic, motor, reward, and stress circuits remains less characterized, despite probable contributions to the motivation to consume alcohol or cognitive dysfunction related to alcohol ingestion (Blaine & Sinha, 2017). Moreover, brain monoaminergic-related neurochemical levels in rodent models following voluntary binge-patterned ethanol ingestion also remains less characterized (Bell et al., 2014(Bell et al., , 2016Ferguson et al., 2019;Iancu et al., 2018). The voluntary ingestion of ethanol at high volumes is an important consideration for understanding the neurobiological underpinnings of the motivation to misuse alcohol. This is a departure from past research, which commonly utilized rodent models of forced ethanol exposure (e.g., gavage, vapor chambers, and i.p. injection) to investigate brain monoaminergic responses to binge-like alcohol drinking (Brown et al., 2000;Gouveia & Hurst, 2013;Stuart & Robinson, 2015). Therefore, even considering the decades of research that has gone into investigating the influence of alcohol abuse on brain monoaminergic activity, much remains to be understood.
The purpose of this study was to evaluate the effects of bingepatterned ethanol drinking on changes in mouse brain and gut monoaminergic activity, using the "Drinking in the Dark" (DID) paradigm.
During the DID procedure, C57BL/6J mice voluntarily consume ethanol to levels exceeding the standard of binge drinking set by the National Institute of Alcohol Abuse and Alcoholism, defined as a BAC of 80 mg% within a 2-hour period (Crabbe et al., 2011;Rhodes et al., 2005). A BAC above this level is sufficient to induce intoxication-like behavior in mice, including ataxia and anxiolysis (Thiele & Navarro, 2014). Male and female mice were considered because sex differences may exist in gut neurochemicals and responses to alcohol ingestion. Therefore, monoamines (i.e., 5-HT, DA, NE), precursor (i.e., l-3,4-dihydroxyphenylalanine (L-DOPA)), and metabolites [i.e., 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxymandelic acid (DHMA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and salsolinol (SAL)] concentrations were measured by ultrahigh-performance liquid chromatography (UHPLC) following the fourth consecutive day of DID. In the gut, neurochemical levels were measured across subregions of the intestinal tract including the duodenum, jejunum, ileum, cecum, proximal and distal colon, and cecal contents. Neurochemical levels were also measured in the liver because little is known about impact of alcohol-related monoamine activity in this organ. Moreover, monoaminergic activity was measured across individual brain areas including the prefrontal cortex, striatum, hypothalamus, cerebellum, brainstem, and remaining cerebral cortex.
These regions of the brain comprise limbic, reward, motor, and HPA pathways that contribute to alcohol misuse and related dysfunction (Uhl et al., 2019). Finally, relationships between monoamine neurochemical signatures across regions of the gut and brain were considered, as this information could provide insight into the possible factors contributing to alcohol-altered communication within the GBA. The results of this study provide detailed information regarding the relationship between voluntary binge-patterned ethanol ingestion and monoaminergic activity in the GBA. These data could have implications for understanding the motivation to misuse alcohol, but also the increased risk of alcohol abuse-related pathological conditions. Moreover, these data also provide insight into sex differences in gut and brain neurochemicals.

Animals
Upon arrival, 24 6-week-old male and female C57BL/6J mice (Jackson Laboratory) were housed individually in standard laboratory cages with a reverse 12-h light/dark cycle (dark at 6 a.m.) and kept at constant room temperature (21 ± 2°C). C57BL/6J mice were incorporated because evidence suggests that this strain voluntarily drinks ethanol to levels exceeding 0.8 mg/ml (0.08 BAC) using the DID paradigm (as described in Rhodes et al., 2005). Mice had ad libitum access to food and water during the entire experiment, except for the DID procedure (see section 2.2 for details). One control male mouse was prematurely removed due to malocclusion. All procedures were approved by the Iowa State University Institutional Animal Care and Use Committee.

Drinking in the dark
Mice began the DID procedure 1 week after arrival. During DID, mice had 2-hour access to graduated sipper tubes containing water (i.e., water group; 6 female, 5 male) or 20% v/v ethanol in water (20% ethanol group; 6 female, 6 male) for 3 consecutive days of acclimation and 4-hour access ±0.25 hours on the fourth day. Four-hour access to sipper tubes has previously shown to yield the highest BEC, which we hypothesized would increase the likelihood of observing neurochemical changes. The DID procedure was initiated 3 hours after the start of the dark cycle, as this coincides with peak consummatory behavior in mice. The amount of fluid ingested was recorded from each sipper tube every 30 minutes to measure drinking patterns over the course of the DID periods. Daily total ethanol consumed was calculated as grams of ethanol per kilogram body weight to control for differences in body mass across mice.

Blood ethanol concentration via gas chromatography
Trunk blood was collected immediately following 4-hour DID access on day 4 for all mice and stored at −80°C. Whole blood was thawed on ice, aliquoted with the ethanol solvent 1,4-dioxane, sonicated, vortexed, and centrifuged (10,000 x g). The supernatant was purified via filtered centrifugation (10,000 x g). Duplicates of purified samples were detected via a gas chromatography-flame ionization detector (GC-FID; Keck Metabolomics Research Laboratory, Iowa State University). To reduce potential ethanol evaporation, samples were kept on ice and recapped quickly throughout the protocol.
Ethanol was not detected in the blood of mice that had access to water.

Tissue preparation and UHPLC analysis
Immediately following DID on the fourth day, mice were decapitated, and brain and gut samples were rapidly collected on a chilled platform simultaneously by separate experimenters. First, a flat edge razor was used to discard the olfactory bulbs and make coronal slice through the brain at approximately 1.97 mm from bregma to collect the area containing the prefrontal cortex. Second, the circle of Willis blood vessels surrounding the exterior of the hypothalamus were discarded via fine point forceps before collecting the hypothalamus (approximate coordinates 0.13 mm, −2.69 anteroposterior from bregma). Third, a coronal slice was taken approximately −0.83 mm from bregma to isolate the striatum. Within this brain slice, the cortex superior and lateral to the corpus callosum was removed from the striatum containing section. Next, the cerebellum was carefully excised. After a mid-sagittal cut, a small flat edge spatula was used to carefully unfold the surrounding cortex and collect the hippocampus. The posterior brainstem area was isolated and collected (see approximate locations −2.69 mm, −8.15 mm from bregma), leaving just the remaining cortical area. For the intestines, an incision was made immediately after the pyloric sphincter and before the ileocecal valve. The duodenum included a 2 cm segment of the proximal end of the small intestine. The jejunum included a 2 cm segment at approximately 15 cm or the middle of the small intestine. The ileum included a 2 cm segment at the distal end of the small intestine.
Intestinal segments between duodenum and jejunum as well as between jejunum and ileum were discarded to remove any ambiguity in discerning the small intestine regions collected. Next, an incision was made on the distal end of the large intestine, the cecum was excised, and entire contents of cecum were collected. A segment of the caudate lobe was excised from the liver. Brain and intestinal regions, liver, and cecal contents were immediately placed into 0.2 M HClO 4 acid and stored at −80°C until UHPLC processing.
Brain, intestinal, liver, and cecal content samples were homogenized using the Omni Bead Ruptor Elite (Omni International) and centrifuged (3000 x g) at 4°C for 30 minutes. After centrifugation, the supernatant from brain samples was aliquoted and stored at −80°C until UHPLC quantification. The remaining gut tissue and cecal samples were further purified and centrifuged (3000 x g) using a 0.45μm filter, aliquoted, and stored at −80°C until UHPLC quantification.
Monoamine quantification was performed in separate batches of brain and gut subregions (to accommodate the large number of samples) using ultra-high-performance liquid chromatography with electrochemical detection (UHPLC-ECD) on a Dionex UltiMate 3000 UHPLC System (Thermo Fisher Scientific). Prior to injection, samples were held in the UHPLC autosampler at 4°C. Neurochemical separation was achieved using Thermo Scientific MD-TM mobile phase at a flow rate of 0.6 ml/min and a Hypersil BDS C18 column (Thermo Scientific). Electrochemical detection was achieved with a 6041RS glassy carbon electrode set to 400 mV.

Statistics
Two-way ANOVA was used to compare the mean volume (ml/kg) consumed between 20% ethanol group (water vs 20% ethanol) and sex (males vs. females) for each day of DID. Additionally, Pearson's correlation coefficients were also used to investigate relationships between BAC and total ethanol consumed on day 4 of DID (see section 2.1).
Concentrations of monoamines and metabolites are expressed as mean μg/g tissue weight ±SEM. Neurotransmitter-to-metabolite ratios (DOPAC/DA, HVA/DA, SAL/DA, DHMA/NE, 5-HIAA/5-HT) were reported as the mean ±SEM for each subregion, as a marker of neurotransmitter turnover. The neurotransmitter ratio DA/L-DOPA mean ±SEM was also reported as a measure of dopamine synthesis.
Data points that exceeded ±2 standard deviations from the mean were removed from the analyses, which can be observed in the degrees of freedom. Mean neurochemical values were compared using 2-way ANOVAs with ethanol treatment (water vs. ethanol) and sex (male vs. female) as between-subject factors. Post hoc analyses were performed using Tukey's HSD following either a significant main effect of ethanol treatment and sex, or interaction between ethanol treatment and sex (see section 3.2 and 3.3). Pearson's correlation was used to follow up on a significant effect of ethanol access to investigate a potentially interesting relationship between BAC and the striatal dopamine metabolites DOPAC and HVA.
Principal component analysis (PCA) was used to identify potentially meaningful trends in the neurochemical data by reducing the large number of variables (regional neurochemical measurements) and incorporating these data into unique, linear combinations called principal components (PCs). PCA is advantageous for visualizing trends in data that has high dimensionality and exhibit collinearity, as is the case for this extensive data set that includes multiple regional neurochemical measurements. The top 2 PCs that explained the most neurochemical variation were used in subsequent analyses (see sections 3.2.4 and 3.4). First, PCA was used to investigate alcohol access and sex-specific differences in neurochemical signatures within the entire gut, as well as regions of the small intestine, and large intestine.
ANOVA was used to follow up on significant group differences among these regional signatures (see section 3.2.4). Second, PCA was performed separately for each brain and gut region to create PCs which represent unique neurochemical signatures for each region collected (see section 3.4). The relationship between the gut and brain PCs as well as the individual neurochemical relationships that followed (Tables 1 and 2) were evaluated using Pearson's correlation with conservative p < 0.01 significance level.

Drinking behavior and blood ethanol concentration
No differences in ethanol ingestion (corrected for body mass) or BAC were observed between male and female mice (see Figure 1A,B). Male mice with water access consumed less fluid compared with the other groups on day 2 of DID ( Figure 1A). On day 3 of DID, mice with ethanol access consumed mildly more fluid compared to mice with water access. On day 4 of DID, there was no difference in drinking behavior between any of the groups. Mean ethanol consumption on day 4 of DID was 9.47 ± 0.41 g/kg and 7.82 ± 0.69 g/kg for female and male mice, respectively. Blood ethanol concentration (BEC) was correlated with ethanol consumed (g/kg) on the fourth day of DID (r = 0.6172, p = 0.0431) ( Figure 1B).

Small intestine subregion
In the duodenum, mouse sex and access to alcohol interacted to influence DA/L-DOPA ratio, F(1, 22) = 6.0604, p = 0.0241 (Figure 2A or large intestine ANOVAs. Taken together, these data may suggest that rodent's sex may influence monoamine neurochemicals in the small intestine to a greater degree than the large intestine, whereas alcohol may influence markers for monoamine activity in the large intestine to a greater degree than the small intestine.

Striatum subregion
Ethanol access had a robust impact on DA-related neurochemicals in the striatum of both male and female mice ( Figure 6A

Gut-brain neurochemical axis
While several lines of evidence suggest the gut and the brain communicate and influence species behavior, the role of monoamines in GBA communication remains less understood. Therefore, the next objective was to investigate potential relationships between neurochemical signatures within gut and the brain, which could provide some insight into communication across the GBA. Because this study collected a large data set summarizing monoamine-related neurochemicals across several gut and brain regions, as well as F I G U R E 8 Neurochemical concentrations and markers of neurotransmitter turnover including the brainstem (A) markers of neurotransmitter turnover and (B) μg/g of neurochemical, the cerebellum (C) markers of neurotransmitter turnover and (D) μg/g of neurochemical, and the hippocampus (E) markers of neurotransmitter turnover and (F) μg/g of neurochemical, +/− SEM. The * represents significant main effect of alcohol; *p < 0.05, **p < 0.01, and ***p < 0.001. Significant main effects of sex are represented as ♂ for males or ♀ for females, each of which indicates which sex had higher values experimental conditions, the data were reduced using PCA in an effort to capture potentially meaningful trends in a manner that can be more easily visualized. First, PCA was performed to reduce neurochemical signatures in each gut and brain region to individual principal components (PCs). Next, Pearson correlation was used to evaluate relationships between gut and brain PCs that contained the most variation in the data (i.e., PC1 and PC2). Two correlations were found below a conservative significance level of p < 0.01, between the jejunum PC2 (representing 28% of the neurochemical variation) and the striatum PC1 (representing 60.7% of the neurochemical variation), and the jejunum PC1 (representing 35.6% of the neurochemical variation) and the cerebellum PC2 (representing 15.5% of the neurochemical variation) ( Table 1). This finding was particularly interesting because past work has indicated a role for the cerebellum in small intestinal motility (Manchanda et al., 1972;Zhu & Wang, 2008), and recent work suggests neural networks originating in the small intestine may contribute to the modulation of monoamine activity in the striatum (Han et al., 2018).
To follow up on the aforementioned relationship between the jejunum with both the cerebellum and striatum, Pearson's correlation was then used to evaluate whether factors like access to ethanol, or sex, drives this relationship. PCA correlations suggest that the relationship between the striatum and jejunum is driven by the mice with water access (Table 1).

DISCUSS ION
Alcohol binge drinking is a prevalent form of substance abuse that can have severe consequences on the body. GI dysfunction has been recently implicated in neurobiological diseases that are also linked with regular alcohol abuse; however, little is currently known about how alcohol abuse influences neurochemicals across the GBA in manners that may be related to the manifestation of disease (Bruce-Keller et al., 2018;Foster & McVey Neufeld, 2013;Lionnet et al., 2018). The influence of voluntary binge-patterned alcohol ingestion on monoamine neurochemical activity across the GBA remains underexplored, despite the known roles of monoamines in both gut and brain function (Mittal et al., 2017). The results of this study provided several key findings related to the influence of ethanol ingestion and sex on monoamine-related neurochemical expressions in mouse gut and brain structures. First, ethanol had a relatively mild effect on gut monoamine-related neurochemical concentrations, primarily influencing intestinal DA activity. Second, some evidence suggests that associations may exist between neurochemical signatures in the small intestine and the striatum, a major component of the brain reward circuit. This finding is consistent with recent work implicating the activity of neural networks in small intestinal with the release of dopamine in the striatum (Han et al., 2018). Third, the DID paradigm yielded some parallels to previous work using forced exposure to high volumes of ethanol, which primarily included augmented dopaminergic activity in striatal and brainstem areas. However, relatively few NE-and 5HT-related changes were observed across brain, outside of the hypothalamus. Finally, several sex-related differences in monoamine-related neurochemicals were observed across gut structures, which could have importance for understanding broader differences in gut function and GI disease between males and females. Together, these data provide insight into the influence of binge-patterned ethanol ingestion and sex on monoamine-related neurochemicals in the GBA.
To the best of our knowledge, this is the first study to investigate the impact of high-volume acute ethanol ingestion on gut monoamine-related neurochemical composition in a rodent model.
One of the most surprising outcomes of this study was that alcohol ingestion only had a relatively mild influence on neurochemical concentrations across the gut (see Figures 2-4), despite mice exceeding NIAAA standards for pharmacologically significant BECs (see Figure 1) (Crabbe et al., 2011;Gilpin & Koob, 2008). This outcome was unanticipated, given evidence suggesting that ethanol can inhibit the activity of enzymes (i.e., alcohol and aldehyde dehydrogenase) involved in the breakdown of all 3 monoamines measured in this study, which could influence the levels of these neurochemicals in the gut (Eisenhofer et al., 2004;Some et al., 2000;Zimatkin et al., 2006). Yet still, neurochemical changes, primarily DA-related, were detected in the gut following alcohol ingestion. A reduction in the ratio of DOPAC to DA concentration was observed across large intestine regions of mice that had access to alcohol during DID (see Figure 3), which may be indicative of an ethanol-related interference with DA turnover . In the small intestine, mice with access to alcohol during DID also displayed a mild increase of jejunal DA (see Figure 2). This finding could be similarly related to alcohol-induced interference with DA metabolism, thereby resulting in greater DA levels, as DA metabolite concentrations in this region were low, failing to reach the detectable limits of UHPLC. While the source of these dopamine-related changes from alcohol ingestion remains unknown, DA is in the GI tract, including multiple bacteria TA B L E 1 Relationship between gut and brain principal components genera, sympathetic and enteric nerves, lamina propria, and epithelial cells, and plays a role in motility, sodium transport, and bicarbonate secretion (Eisenhofer et al., 1997;Mezey et al., 1996). Therefore, alterations in dopamine activity in the gut following episodes of alcohol abuse could influence gut physiology and activity across the GBA.
Research is still uncovering the functions of DA in the gut; therefore, it is not entirely clear how reduced DA metabolism and higher DA levels might influence GI or GBA function. Dopaminergic enteric nerves have essential functions in slowing gastrointestinal motility (Rao & Gershon, 2016), which may be one of the contributing factors to decreased motility from ethanol consumption. Intestinal dopamine also has a homeostatic role in regulating electrolytes via in high salt diets by inhibiting Na+-/K+-ATPase activity in the jejunum (Vieira-Coelho et al., 2000). Thus, elevated jejunal DA may be a compensatory response to maintain electrolyte balance during alcohol ingestion. Additionally, DA released from nonneuronal sources such as gastric and pancreatic juices may be protective against mucosal injury in the proximal small intestine (Magro et al., 2002). Thus, a possibility remains that toxic metabolites related to alcohol metabolism (e.g., acetaldehyde) could be facilitating nonneuronal DA release, resulting in a protective increase of DA levels in the jejunum.
While the sources and impact of alcohol ingestion-induced changes to DA remain clear, changes to the concentration of favoring greater levels of DA could significantly influence GI physiology. These topics warrant a more detailed investigation in the future as it could be important for a more complete understanding of GI pathophysiology and more efficacious treatments.
A relationship was observed between neurochemical signatures in the jejunum and striatum of mice with water, but not ethanol, access (see Table 1). Taken in the context of other recent findings, this relationship may provide new insight into the monoaminergic influences on GBA communication. Indeed, the optogenetic stimulation of neural networks located in the small intestine has been shown to potentiate the release of dopamine in the striatum (Han et al., 2018), a critical component of the brain's natural reward circuit. In the current study, the absence of relationship between small intestine and striatum neurochemicals of mice with alcohol access could provide novel evidence that ethanol ingestion might disrupt synchronous patterns of neural activity across the GBA. Monoamines in the intestine have been shown to mediate GI sensory information to afferent sensory nerve fibers (Bellono et al., 2017;Kaelberer et al., 2018;Martin et al., 2018). Therefore, it is possible that changes in luminal contents either directly from ethanol or indirectly (e.g., inflammation, bacterial changes, and bacterial by-products) affect the sensory signals being relayed to the striatum. However, ethanol has a potent influence directly on brain neurochemicals, which makes it difficult to disentangle the influence of intestinal modulators.
Nonetheless, a potential alcohol-induced desynchronization of the coordinated communication between the gut and the brain could underlie the increased the risk of disease, for example, in Parkinson's disease and depression (Foster & McVey Neufeld, 2013;Gorky & Schwaber, 2016;Klingelhoefer & Reichmann, 2015). Our data, taken together with previous work, provide new evidence suggesting synchronized neurochemical activity may exist between the small intestine and the brain's natural reward circuit. The potential role for monoamines in coordinated communication between the gut and brain, as well as how ethanol ingestion may alter the GBA, warrants a more detailed investigation in future studies. in outcomes from these models with regard to levels of 5HT and NE (Das et al., 2016;De Witte, 1996;Gongwer et al., 1989;Milio & Hadfield, 1992;Murphy et al., 1988;Nurmi et al., 1994;Pohorecky & Jaffe, 1975), the preponderance of evidence suggests a role for the mesolimbic dopaminergic system (i.e., ventral tegmental area and nucleus accumbens) in the acute reinforcing effects of alcohol (Koob, 2014). The current DID paradigm parallels previous findings that elevated dopaminergic activity in brain reward structures following binge ethanol administration (Barbaccia et al., 1982;Dar & Wooles, 1984;Fadda et al., 1989;Honkanen et al., 1994;Imperato & Di Chiara, 1986;Murphy et al., 1988;Nurmi et al., 1994), while also observing fewer and less robust brain-wide monoaminergic responses outside of the hypothalamus (see Figures 6-8). Indeed, access to ethanol increased concentrations of DOPAC and HVA in the striatum area (see Figure 6), suggesting ethanol augments activity in the DA reward pathway. Consistent with other studies, the DA metabolite SAL was also present in the brain regions that contain prevalent DA pathways, including areas like the striatum and brainstem. In these regions, elevated SAL activity has received some attention for its possible role in linking the motivation to ingest high volumes of alcohol and neurodegeneration (Kurnik-Łucka et al., 2018;Peana et al., 2016). Given that SAL is a product of DA and acetaldehyde, it could be hypothesized that an increase in the ratio of SAL to DA might also be observed in the brainstem or striatum following a period of alcohol access. Contrary to expectations, the ratio of SAL to DA was actually lower in the striatum and unchanged in the brainstem following ethanol access (see Figures 6 and 8).
However, the conditions that lead to the augmented SAL production remain poorly understood and may be more than just a consequence of alcohol abuse, also involving exogenous or endogenous sources of dysregulation within catecholamine enzymatic pathways (Kurnik-Łucka et al., 2018). Recent in vivo work found that SAL production was most stimulated following the intraperitoneal administration of high volumes of ethanol and L-DOPA in combination with a cocktail of inhibitors of enzymes involved in the breakdown catecholamines (Boston, 2016). Taken together, the current data underscore the importance of using rodent models of voluntary binge-patterned ethanol drinking (e.g., DID) to gain a more complete understanding of the acute effects that alcohol abuse has on brain function.
While not initially the primary focus of this study, several interesting sex-specific neurochemical responses were observed across the gut (see Figure 5). In fact, data collected from PCA suggested the sex differences in neurochemical concentrations may be pronounced in the small intestine (see Figure 5B). This finding adds to a body of growing literature suggesting the small intestine many contain a more robust sexually dimorphic neurochemical response than the large intestine. For instance, differential gene expression in the small and large intestine of mice found the majority of sexually dimorphic genes in the small intestine compared to the large intestine, and many of these genes were involved in regulating neurotransmission (Steegenga et al., 2014). Consistent with this observation, monoamine synthesis enzymes (i.e., L-amino acid decarboxylase) were found in higher concentrations in the small intestine, but not the colon, of male compared to female mice (López-Contreras et al., 2008). Thus, this could help explain differences in monoamine concentrations or turnover, like those observed in the current study (see Figure 2). Additionally, several studies have reported sex differences in the microbiome (de la Cuesta-Zuluaga et al., 2019;Hormonedependent et al., 2013;Kim et al., 2020;Vemuri et al., 2019), which could be relevant because some microbial species synthesize 5-HT and DA in the gut (Galland, 2014). Sex differences in monoaminesynthesizing enzymes and the microbiome could account from at least some of the sex differences (less L-DOPA in the female jejunum and ileum, less 5-HIAA in the ileum, greater levels of NE turnover in the jejunum, and more DA and 5HT in the large intestine cecal contents) observed in the current study (see Figures 2 and 3). Taken together, these data may provide some insight for understanding sex-biased GI function or dysfunction. For instance, sex differences found in GI monoaminergic systems may contribute to the variation in treatment efficacy between males and females with irritable bowel syndrome (Viramontes et al., 2001). However, monoaminergic sex differences in the intestinal tract have not been well characterized, and consequently, the potential functions currently remain less clear should be followed up with more detail in future work.
Furthermore, sex differences in liver 5HT concentrations following ethanol ingestion in the current study could have relevance for understanding susceptibility to liver damage. Indeed, the liver is the body's main source of alcohol metabolism and can become acutely damaged following ingestion of ethanol. In the current study, female mice with access to ethanol displayed higher concentrations of liver 5HT than male mice (see Figure 4). Female sexspecific hormones, such as estradiol, reduce the activity of 5HT reuptake transporters and degradation enzymes (Benmansour et al., 2012;Koldzic-Zivanovic et al., 2004). Therefore, it is possible that alcohol-stimulated 5HT activity in the liver will be less likely to clear in females than males, thereby leading to greater tissue concentrations of 5HT in the female liver (see Figure 4). This potential outcome is particularly noteworthy because evidence indicates that livers of women who drink alcohol excessively may be more prone to damage than males. In rodent models, females are more likely to display alcohol-induced liver steatosis following a single episode of high-volume alcohol exposure, a process dependent on serotonin receptor activity (Pang et al., 2020;Wagnerberger et al., 2013). Taken together, sex-related hormones may be driving the ethanol-induced increase in 5HT concentrations in a manner that contributes to female susceptibility to alcohol-related liver injury.
Future studies could investigate this topic as it could have importance for the etiology of liver disease in women that struggle with alcohol use disorder.
The present study added several new perspectives furthering our understanding on influence of alcohol binge-like drinking and sex on GBA physiology. Alcohol's influence on monoamine concentrations in the gut and brain could play key roles in altering communication across the GBA in manners that may contribute to the development, or severity, of neurobiological and intestinal diseases. Moreover, potential sexual dimorphism of monoaminergic activity within the gut could have key implications for reported differences in gut functions between males and females, as well as sex-specific treatments for intestinal disorders known to involve abnormal monoamine activity. Together, these findings lay the foundation for understanding the role of monoamines in GI function, GBA communication, and provide insight into targeted interventions (based on diet, lifestyle, sex, etc.) for diseases associated with alcohol misuse and altered GBA communication.
The functional implications of GBA monoaminergic-related neurochemical differences due to alcohol or mouse sex should be topics for future research.

ACK N OWLED G EM ENTS
We would like to thank Dr. Lucas Showman from W.M. Keck Metabolomics Research Laboratory at Iowa State for assisting with analysis of blood ethanol concentration via gas chromatography.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.