Effects of moderate alcohol consumption on behavior and neural systems of Wistar rats

: Chronic alcohol consumption affects various neurotransmitters, especially those implicated in the transitioning to alcohol use disorders (particularly dopaminergic and CRFergic systems). Few studies have investigated moderate alcohol consumption and its harmful consequences. The objective of this work was to analyze behavioral and neurochemical (dopaminergic and CRFergic systems) alterations during chronic moderate alcohol consumption. Twelve male Wistar rats were submitted to an intermittent alcohol ingestion protocol (alcohol group) for four weeks. The control group consisted of six rats. Open Field and Elevated Plus Maze tests were used for analysis of motor and anxiety-like behaviors. Immunohistochemistry analysis was performed in dopaminergic and CRFergic systems. Animals exposed to alcohol consumed moderate doses, chronic and intermittently. Behavioral tests detected fewer fecal boli in the alcohol exposed group, and immunohistochemical analysis indicated fewer dopamine-immunoreactive cells in the ventral tegmental area, and more CRF-immunoreactive cells in the anterior cingulate cortex and dorsolateral septum in this group. Thus we concluded that Wistar rats that consumed moderate doses of alcohol voluntarily and chronically showed a discreet anxiolytic effect in behavior, and a hypodopaminergic and hyperCRFergic neurochemical condition, which together are strong inducers of alcohol consumption predisposing to the development of alcohol use disorder (AUD).


INTRODUCTION
According to the World Health Organization (World Health Organization 2018), 43% of the world population is currently drinkers (drink any amount of alcohol).Heavy episodic drinking (more than 60 grams of pure alcohol, or 6 standard alcoholic drinks, on one occasion or at least once a month) was prevalent in 18.2% of the world population, and 39.5% among drinkers in 2018(World Health Organization 2018).This consumption pattern is related to extensive damage to the organism (Rehm et al. 2010).For a long time, low or moderate doses were seen as not-harmful or even protective in certain conditions (Lichtenstein et al. 2006), although recent research indicated that there is not a clear threshold between safe and harmful alcohol ingestion.Alcohol consumption even at low doses, accordingly to Global Burden of Disease Study 2016(GBD 2016Alcohol Collaborators 2018), can induce deleterious consequences to the organism (Amodeo et al. 2017, Burton & Sheron 2018).
Alcohol effects in the brain involve several neurotransmitter systems (Roberto & Varodayan 2017).The initial pleasurable response (positive reinforcement by consumption; stimulatory effect) is orchestrated mainly by the dopaminergic system through the activation of the mesolimbocortical pathway that comprises the ventral tegmental area (VTA), nucleus accumbens (NAc), and distinct regions of the prefrontal cortex (PFC).Additionally, alcohol can help to cope with negative emotions (i.e.anxiety, dysphoria, etc.) that arise from environmental stress or periods of heavy alcohol withdrawal (negative reinforcement by consumption; anxiolytic effect).In this case, alcohol acts on the stress response system, which has the corticotrophin release factor (CRF) peptide as its main activator.Consequently, disbalances on the CRFergic system are critical to the emergence of alcohol use disorders (AUD) (Heilig 2007, Koob et al. 2014, Koob & Volkow 2016).
The dopaminergic superstimulation during alcohol consumption can promote impairments of the system after prolonged use and prompt compulsive alcohol consumption (Söderpalm & Ericson 2011).Moreover, the activation of CRF system by alcohol consumption can be trigered acutely and chronically, having long term effects (Allen et al. 2011).CRF activates the hypothalamic-pituitary-adrenal axis (classically known as the HPA axis) for the neuroendocrine stress response.But beyond that, different areas with extra-hypothalamic CRFergic neurons seem to contribute to behavioral and mental responses to stressors.Some of these areas have been linked to alcohol consumption such as the lateral septum (responsible for emotional reactivity), the amygdala (one of the centers of the stress response that is associated with the intensification of consumption), the bed nucleus of stria terminalis (BNST), and portions of PFC, such as the anterior cingulate cortex (ACC) (both involved in relapse) (Wscieklica et al. 2016).
The literature still lacks studies investigating low to moderate alcohol consumption.Therefore, to explore the effects of such doses of alcohol is important because the misconception that moderate drinking does not have detrimental consequences to the organism persists in society.The identification of biological effects during moderate drinking can help the development of preventive strategies before the subject advances to heavier drinking and AUD onset.Hence, we hypothesize that drinking moderate doses of alcohol could induce subtle alterations in brain circuits involved in posterior complications such as AUD.Thus, the objective of this work was to evaluate behavioral and neurophysiological responses in the dopaminergic and CRFergic systems during moderate chronic alcohol consumption in an animal model.

Subjects
Eighteen male Wistar rats (CEDEME -Centro de Desenvolvimento de Modelos Experimentais para Biologia e Medicina, Universidade Federal de São Paulo -UNIFESP, Brazil), about 60 days old, weighing 240-310g, were kept under controlled environmental conditions (21 ± 1°C, dark-light cycle -12h on-off cycle, 7:00 AM to 7:00 PM lights on) with food and water ad libitum.The experimental protocols were developed according to principles adopted by Brazilian Society of Neuroscience and Behavior, which are based on the conditions stated in "Guide for the Care and Use of Laboratory Animals" (Institute of Laboratory Animal Resources 1996) and approved by UNIFESP Research Ethics Committee (number 3482080218).
Animals were sorted into one per cage (for precise liquid ingestion control) in translucent cages side by side constituting the following groups: Control group (CO): six rats were not subjected to alcohol ingestion, comprising the control group.Measurements of body mass,  (2022) 94(3) e20210673 3 | 13 water, and food ingestion were coincident with the other (experimental) group.
Alcohol group (AL): twelve rats had intermittent access to ethanol solution (20%, v/v) with the two-bottle choice paradigm (Carnicella et al. 2014, Wscieklica et al. 2016) on Mondays, Wednesdays, and Fridays during a 24h period (12h light and 12h dark cycle) during 4 weeks.The placement of alcohol bottle in the cage was switched with water bottle in every session, thus avoiding the influence of placement bias.Between sections of alcohol availability, two bottles of water were offered per cage.Fluids were administered in 200ml bottles with hermetically sealed lids, tightly secured on the cage walls.The difference in the sample sizes is to account for the variability of drinking behavior (Momeni & Roman 2014).The administration method is illustrated in Figure 1.
To control the aforementioned variables, every week (on Mondays and Fridays) each animal was weighed.Food ingestion was controlled by offering a known amount of food in the beginning of the week and weighing the residual at the end of the week.Water and alcohol were measured daily by weighing, before and after their administration.Solutions (alcohol and water) were double-checked by gauging its volumes also before and after their administration.

Procedures
Behavioral Tests -Open Field (OF) The Open Field (OF) test was performed on a round arena (60 cm of diameter), with the floor radially divided in inner and outer sections and also divided into 12 equal parts, its walls were 50 cm high, the apparatus was made of plexiglass.Tests were performed under 60 lux illumination and the apparatus was located in a separate room.Animals were briefly habituated to the testing room, out of the apparatus, for about 10 minutes before the test, subsequently, each animal was placed in the center of the apparatus for testing.
All the animals were subjected to OF in the morning (8:00 AM to 11:00 AM) after the last hours of the last session of the alcohol administration protocol.The test was performed to access motor parameters (line crossing) allowing inferences on anxiety-like behaviors, such as if the animals were more active in the center or the periphery of the arena (the latter related to anxiety-like behavior), as well as the largest number of fecal boli (physiological parameter), rearing (standing on their hind legs, indicating vertical exploratory activity), and grooming (cleaning their own body, a stereotypical behavior) represent a greater degree of anxiety (Kliethermes 2005).The procedures were videorecorded and later assessed by a researcher blind to the assigned group of each rat.

-Elevated Plus Maze (EPM)
After the motor activity test, all animals were submitted to Elevated Plus Maze (EPM) test to evaluate the possible effects of chronic alcohol ingestion on the anxiety state of the animals and its relation to stress-related brain areas (Kliethermes 2005).The EPM is based on the principle that the animal avoids open areas and the confinement in the closed arms of the maze can be related to a state of anxiety.Therefore, the overall exploration of the maze was analyzed by the following behaviors.Number of entries and time spent on the closed or open arms, look outs (projecting only the head out of the maze arm), transitions between the arms, rearing, head dips (dipping of the head below the level maze floor), and grooming, (Pellow et al. 1985).The apparatus was located in another similar room where animals underwent 10 minutes of habituation before the test.At the beginning of the test each animal was placed in the center of the apparatus that was made of wood, painted in white, and each arm had the same dimensions (50 cm x 12 cm), both of the arms were enclosed by a 40 cm high wall.
In both tests, that occurred after the last session of alcohol exposure, each animal was evaluated for 5 minutes and entered the respective apparatus in random order, and after each animal finished the test the apparatuses were cleaned with alcohol 70% v.v.The procedures were videorecorded and later assessed by a researcher blind to the assigned group of each rat.

Anesthesia, Blood-Analysis, Perfusion, and Microtomy
Each animal, immediately after the behavioral test, was perfused in accordance with the protocol of Wscieklica et al. (2019).This procedure was performed after the last session of alcohol exposure.The animals were anesthetized by intraperitoneal injection of a dose of 1 ml/kg of ketamine (40 mg/kg), xylazine (20 mg/kg), fentanyl (0.3 to 0.5 mg/kg) and acepromazine (1ml/kg) mixture.Immediately before the perfusion, blood samples were collected from the heart of the animals (left ventricle) utilizing a syringe and transferring the blood samples to heparinized tubes.The blood alcohol concentration (BAC) was analyzed utilizing the Multiple Reagent Test NAH-ADH (Sigma Aldrich, EUA), exactly as described by Conte et al. ( 2019), hence assessing the alcohol availability in the blood.Subsequently, perfusion was performed with a 4% paraformaldehyde solution (from formaldehyde heated to 60-65 °C) in H 2 O at 4°C, pH 9.5, for approximately 25 min.The brains were post-fixed for 1h in the same fixative solution and then stored in a solution of 30% sucrose for cryoprotection at 4 °C.Regularly sampled series (1 in 5) of 30µm-thick frozen sections were cut in the coronal plane and stored in ethylene glycolbased cryoprotectant at -20 °C.

Immunohistochemistry for Dopamine or CRF
To investigate immunoreactivity to dopamine, in its main producing area, the mesolimbocortical system, the ventral tegmental area (VTA; bregma: -5.04) was analyzed (Dahlstroem & Fuxe 1964).For analysis of immunoreactivity to CRF the main brain areas associated with emotional stress responses in the extra-hypothalamic system were analyzed: the amygdala and its divisions (central, medial and basolateral; bregma: -2.76); bed nucleus of stria terminalis (BNST) (bregma: -0.60); lateral septum and its divisions (dorsal, intermediate, and ventral; bregma: +0.60); and anterior cingulate cortex (ACC) (bregma: +0.60) (Sawchenko et al. 2010).The ACC was used as a representative area of PFC in stress response since functional alterations in this area are a predictor of relapses in alcohol consumption (Zakiniaeiz et al. 2016).
Brain sections were subsequently submitted to anti-dopamine or anti-CRF immunohistochemistry protocol based on Wscieklica et al. (2016).Slices were incubated with anti-dopamine polyclonal antibody (rabbit; 1:500; MyBioSource; catalog number: 2007234, San Diego, USA), or with anti-CRF polyclonal (rabbit; 1:1000; Bioss, catalog number: bs-0246R-HRP, Massachusetts, USA).The slices were rinsed in a solution with hydrogen peroxide (0.3%; Sigma, St. Louis, MO, USA) before incubation with the primary antibody, to avoid interference of endogenous peroxidase.Tissue incubation with the primary antibody occurred in Triton-X solution to increase neuronal membrane permeability and normal goat serum to avoid nonspecific binding.In sequence, the slices were incubated with biotinylated anti-rabbit secondary antibody (goat; 1:1000; Vector Laboratories, Burlingame, CA, USA).Avidin-biotin (Vectastain Elite Vector Laboratories, Burlingame, CA, USA) were added and finally, the diaminobenzidine chromogen (DAB; 0.05%; Sigma) was applied to the tissue and amplified using nickel ammonium sulfate.After the immunoperoxidase, the tissue was mounted on gelatin-coated glass slides and dehydrated for microscopy.
Results were analyzed with a Zeiss -Axio Observer D1 microscope.Dopamine or CRF positive cells were blind analyzed utilizing Image Pro-Plus™ v. 6.0 software.The color spectrum utilized in the measurements was adjusted from dark brown to black and a fixed bi-dimensional area (approximately 500µm x 500µm) was adopted for each region of interest.

Statistical analysis
Group analyses of quantitative results were performed with the unequal variance t-test (Welch´s test) as recommended by Ruxton (2006), as it accounts for unequal variances and is equally robust as a Student-t test when variances are equal, or one-way or two-way analysis of variance (ANOVA).Whenever the normality assumption was violated, the Mann Whitney U test (or the appropriate correction) was performed (software JAMOVI v.1.2).P values lower than 0.05 (p<0.05) were considered statistically significant.Results are reported as mean ± standard deviation (SD) and effect size when applicable (Cohen´s d or eta-square).

RESULTS
Alcohol, blood alcohol concentration, food, water ingestion, and body weight The mean alcohol consumption during the experimental period of the alcohol group was: 2.89 ± 1.15 g/kg/24h.The sphericity assumption was violated as indicated by Mauchly's test (p = 0.045); thus, the repeated measures one-way ANOVA underwent the Greenhouse-Geisser correction to verify if there were significant differences along time.No statistically significant variation along time was observed F (1.85, 20.34) = 0.37; p = 0.67; η 2 = 0.01.
Regarding the mean blood alcohol concentration, it was observed 77.70 ± 28.30 mg/ dl in the AL group.
Concerning food intake during the experiment, the average consumption (per week) of CO and AL groups were, respectively: 199.75 ± 24.90 g, and 187.75 ± 23.42 g.Repeated measures two-way ANOVA only indicated an effect of time [F (3, 48) = 11.08;p < 0.001; η 2 : 0.25].Tukey posthoc indicated differences along all the weeks (p < 0.05), although no particular trend was observed.

Behavioral tests: Open Field (OF) and Elevated Plus Maze (EPM)
Based on behavioral tests performed, the only statistically significant result was regarding the number of fecal boli during the OF test [CO group: 7.17 ± 3.19; AL group: 2.58 ± 2.43; Welch T (8.02) = 3.09; p = 0.01; d = 1.70].The other parameters analyzed by these tests, such as stereotypical behaviors, were not statistically significant, as shown in Table I.

Dopamine and CRF immunoreactivity
Data from immunohistochemical analysis from dopaminergic neurons (dopamine-ir) of VTA, indicated that AL group showed significantly less immunoreactive cells than animals of CO group [528.00 ± 246.00 and 893.00 ± 341.00 respectively; Welch T (7.71) = 2.33; p = 0.04; d = 1.31], as can be observed in Figure 2.
Regarding the immunoreactive cells to CRF (CRF-ir) a significant increase was detected in the AL group when compared to CO group in the anterior cingulate cortex; CO: 226.00 ± 29.00, and AL: 567.00 ± 225.00; as Shapiro-Wilk test indicated a violation of normality (p = 0.001), Mann-Whitney U test was performed, U = 0; p = 0.002; d = -1.78.Another region with similar result was the dorsolateral septum; CO: 176.0 ± 27.2, AL: 293.0 + 121.0;Welch T (13.29) = -3.17;p = 0.007; d = -1.12.These are shown in Figure 3.These analyses were performed with a control group sample of 5 due to mechanical damage during the immunohistochemical process.

DISCUSSION
In this model of non-invasive chronic alcohol intake, we aim to mimic alcohol consumption in moderate doses using a voluntary alcohol ingestion protocol in Wistar rats that are genetically heterogeneous and typically do not drink large amounts of alcohol (Palm et al. 2011).The animals consumed a stable and moderate amount of alcohol, then to exclude any animal due to high drinking behavior was not necessary (Leeman et al. 2010).The animals showed slightly higher levels of alcohol in the blood compared to other studies, an effect that can be explained by the fact that blood collection occurred at the end of the dark cycle, the phase of greatest consumption, and also by the ofteninconsistent nature of these analyzes (Crabbe et al. 2011, Dilley et al. 2018).However, the mean BAC of 77.07 mg/dl (0.07%), (and consequently 2.89 g/Kg/24h of ingested alcohol) is comparable to the blood alcohol level considered legal for humans to drive in various countries.This BAC level, in humans, is related to euphoria, loss of inhibition and impairment of some motor skills (Dasgupta 2017).Therefore, alcohol intake levels and blood availability achieved in the current study are similar to those pursued by humans that drank moderately and avoided intoxication.
The food and water consumption, as well as animal weight, did not differ between the groups indicating any effect of alcohol consumption in these parameters.Although the differences in time were significant, they were expected.Also, the behavioral tests indicated fewer fecal boli in the alcohol group, which can be interpreted as an anxiolytic effect (Archer 1973).Alcohol has anxiolytic properties and all analyses were performed with animals under the chronic effects of the substance and not during the withdrawal phase as confirmed by the BAC levels.Motor behavior showed no difference between groups, which is in line with the defecation result of a mild anxiolytic effect (Lister 1990, Wegener et al. 1991, Palasciano et al. 1995).The other behavioral parameters could not detect any further alterations.
The mesocorticolimbic system undergo adaptative alterations after chronic alcohol exposure.During the ingestion of low or high doses of alcohol, there is an acute increase in the expression of dopamine in VTA (You et al. 2018), while chronic and intermittent high consumption (10/g/kg/day) can lead to decreased neuronal activity in VTA 3 to 6 weeks after the end of treatment (Shen et al. 2007), which is in accordance with our results, although our data indicate this effect with moderate consumption.Therefore, the reduced dopaminergic activity in this region may be responsible for the transition to AUD, since the drug could be utilized to attenuate negative effects of unpleasant stimuli, thus coping with lower dopamine levels (Koob et al. 2014, Nutt et al. 2015).It seems that the dopaminergic system suffers a tolerance or overload effect after the initial stages of consumption even when consuming moderate doses, culminating to blunted levels of neurotransmitter.Thus, we can understand that after the acute phase, chronic intake leads to a hypodopaminergic condition, even, as we have shown in this study, in moderate doses.But this phenomenon requires further investigation (Charlet et al. 2011).However, it appears that the hypodopaminergic condition can promote the maintenance of alcohol consumption, even in moderate doses.
Studies show that activation of CRF system occurs during alcohol consumption, not only during withdrawal and during its adverse consequences (Quadros et al. 2016).Generally, alcohol seeking behaviors are induced, partly, by the activity of CRF system in specific regions of the brain involved in cognition and emotional control (Anthenelli 2012, Blaine & Sinha 2017).In this study, the CRFergic immunoreactivity was significantly higher in the anterior cingulate cortex of the alcohol group.This region is responsible for the integration of various circuits to perform executive functions, such as controlling impulsive behaviors (Vassena et al. 2017).It is particularly affected by alcohol consumption (Yang et al. 2016), as shown by recent work from our group, where we reported a volume reduction of this area in rats that consumed alcohol in high doses (Conte et al. 2019).This effect may be associated with the hyperregulation of stress related neurons of this area that in general has a degenerative effect on the nervous system.There is a considerable distribution of CRF receptors (Potter et al. 1994)  The dorsolateral septum also presented similar results to ACC.This region (lateral septum) is especially important in relation to affective and motivated behaviors, as it functions as a center for the integration of sensory and behavioral stimuli from cortical regions, amygdala, hippocampus, hypothalamus and mesocortical dopaminergic system, among others.Its role in anxiety, fear, mood, and substance use disorders are intricate (Sheehan et al. 2004) and there are few studies with this particular region, especially involving CRF system and substance use.The higher amount of immunoreactive CRF cells in this region is interesting as animals were ingesting alcohol voluntarily, and the region regulates motivational, contextual, and stress behaviors (Besnard et al. 2019).Conversely, the hyperactivation observed may be involved in drug seeking behavior, not only for the stressful effects of abstinence since our protocol is of chronic drug supply (Calfa et al. 2006, Singewald et al. 2011, Scalize Hirata et al. 2019), but by the drug effect itself in an area involved with motivated behaviors.This specific region also has shown responsiveness to anti-depressant treatments (Sheehan et al. 2004) indicating a role in mood control, which is related to alcohol consumption.The lack of studies about the regions made it difficult to come to further conclusions.
Limitations of this study include the use of only male rats, which prevented the evaluation of these effects on females.There was no temporal analysis performed, as this study was cross-sectional, and longitudinal studies are encouraged to address the progressive effects of moderate doses of alcohol.Also, analyzing different levels of alcohol voluntarily consumed may help to understand how changes in the brain dopaminergic and CRFergic systems are related to the amount of alcohol ingested.

CONCLUSIONS
Through this study, we concluded that Wistar rats that consumed moderate doses of alcohol voluntarily and chronically showed a discreet anxiolytic effect in behavior, and a hypodopaminergic and hyperCRFergic neurochemical condition, which together are strong inducers of alcohol consumption predisposing to the development of alcohol use disorder (AUD).
RAFAEL CONTE et al.ALCOHOL EFFECTS ON BEHAVIOR AND NEURAL SYSTEMSAn Acad Bras Cienc

Figure 1 .
Figure 1.Representation of the alcohol ingestion protocol.

Table I .
Parameters analyzed by the Open Field test and Elevated Plus Maze, in Control (CO) and Alcohol (AL) groups.

Table II .
Number of immunoreactive cells to dopamine and CRF in the analyzed brain areas in Control (CO), and Alcohol (AL) groups.