Jérôme Jeanblanc

Benjamin Rolland
email: benjamin.rolland@univ-lyon1.fr

Fabien Gierski
email: fabien.gierski@univ-reims.fr

Margaret P Martinetti
email: martinet@tcnj.edu

Mickaël Naassila
email: mickael.naassila@inserm.fr

Mickael Naassila
email: mickael.naassila@inserm.frjerome.jeanblanc@u-picardie.fr

Prof. Mickael Naassila

Animal models of binge drinking, current challenges to improve face validity

Keywords: binge drinking, animal models, rodents, validity, alcohol

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Definition and diagnostic of BD in humans

Binge drinking (BD) refers to a specific pattern of alcohol use, namely drinking large amounts of alcohol in a short period of time [START_REF] Courtney | Binge drinking in young adults: Data, definitions, and determinants[END_REF]. The BD behavior is characterized by the intensity and also by the frequency of the drinking episodes and BD behavior is thus including longer or shorter periods of no consumption (between binges) (SAMHSA, 2016).These periods of abstinence, among other criteria such as the loss of control over alcohol consumption and the compulsion, allow the distinction between BD and some other patterns of unhealthy alcohol use, especially severe alcohol dependence (Rolland and Naassila, 2017) in which daily alcohol consumption is usually observed. For this reason, BD is frequently defined as an "episodic heavy use of alcohol" (WHO, 2014;SAMHSA, 2016).

In the epidemiological literature, the criteria for BD include exceeding drinking thresholds in a short amount of time. For example, the World Health Organization (WHO) defines BD as the "consumption of 60 or more grams of pure alcohol (6+ standard-drinks in most countries) on at least one single occasion at least monthly" (WHO, 2014). However, the National Institute on Alcoholism and Alcohol Abuse (NIAAA) defines BD as "a pattern of drinking alcohol that brings blood alcohol concentrations to 0.08 gram percent or above. For the typical adult, this pattern corresponds to consuming five or more drinks (male) [American drinks, i.e., 14g of ethanol], or four or more drinks (female), in about two hours" (NIAAA, 2017, [START_REF] Courtney | Binge drinking in young adults: Data, definitions, and determinants[END_REF]). Compared with the WHO definition, the NIAAA definition of BD also involves a minimum drinking speed and deliberately seeking drunkenness. Consequently, these two common definitions of BD convey subtle conceptual differences, and the epidemiological populations that they delineate do not display the same clinical profile and severity level (Rolland et al., 2017).

Moreover, the typical definitions of BD do not explore the frequencies of drinking or heavy drinking behaviors, or the average amount of alcohol used during BD episodes but rather focus on the thresholds described in the different definitions and thus define BD groups (above the threshold) and non BD groups (below the threshold). As the severity of BD can directly depend on these parameters, BD populations studied using the usual definitions only are actually very heterogeneous, which raises some important scientific concerns (Ceballos andBabor, 2017, Rolland andNaassila, 2017). In this regard, a recent study has shown that, compared with the WHO criteria, the NIAAA criteria for BD identifies individuals with more severe drinking patterns and alcohol consequences (Rolland et al., 2017).

Taking a mere drinking threshold to define such a complex behavior like BD can lead to the undescreening people that are drinking with an intensity that is very far from this threshold and thus display very specific and important characteristics (regarding drinking behavior and personality). To this end, in its recent newsletter (NIAAA Spectrum, 2017) the NIAAA refers to the study of [START_REF] Hingson | Drinking Beyond the Binge Threshold: Predictors, Consequences, and Changes in the U.S[END_REF] that proposes new cut-offs of ethanol consumed in a single occasion (for women and men, respectively): level I (56-98g and 70-126g), level , and level III (>168g and >210g). They also defined levels II and III (i.e., exceeding 112g and 140g in a single occasion for women and men, respectively) as "extreme binge drinking."

Although these thresholds account for the heterogeneity of the behavior, they do not address the frequency of the behavior. For example, an individual who has had one panic attack in the past month may indeed have panic disorder, but the recurrence of attacks (as well as other criteria) are needed for the diagnosis of this disorder.

Therefore, valid definitions of BD require not only specific features of the drinking patterns, such as drinking intensity per drinking occasion, but also frequency of BD behaviors and periods of abstinence between binging episodes. Moreover, recent data suggest that BD is a heterogeneous phenotype. Consequently, binge drinkers should not be considered as a unitary group, but rather as a heterogeneous population of individuals displaying particular gender and personality dimensions [START_REF] Gierski | Cloninger's Temperament and Character Dimensions of Personality and Binge Drinking Among College Students[END_REF], as well differences in drinking motives and impulsivity (Lannoy et al., 2017a).

In an attempt to provide a more behavioral definition of BD, [START_REF] Townshend | Patterns of alcohol drinking in a population of young social drinkers: a comparison of questionnaire and diary measures[END_REF] proposed the computation of a "binge score," which is based on both the frequency of excessive consumption and the average speed of consumption (Townshend andDuka, 2002, Smith et al., 2017). In one application of this measure, participants with a history of BD had more severe brain damage and cognitive impairment compared with social drinkers, and the degree of these impairments was positively correlated with the binge score [START_REF] Smith | Altered white matter integrity in whole brain and segments of corpus callosum, in young social drinkers with binge drinking pattern[END_REF].. Such findings revealed that both the level of intoxication and drinking speed (producing higher binge score) were crucial factors in the impact of BD behavior [START_REF] Smith | Altered white matter integrity in whole brain and segments of corpus callosum, in young social drinkers with binge drinking pattern[END_REF]. In particular, fast alcohol consumption, especially on an empty stomach, allows ethanol to reach the blood more rapidly, and increases blood alcohol levels with a life-threatening speed. "Happy hours", during which alcoholic drinks are cheaper for a limited period of time, dramatically promote BD behavior and severe intoxication [START_REF] Thombs | Drink specials and the intoxication levels of patrons exiting college bars[END_REF], and new policies have emerged to curb BD. Inspired by human anecdotal evidence and the culture of "happy hours," animal models have evolved to mimic BD by shortening the access to alcohol.

Animal models are meant to parallel the human condition; however, rodents do not readily consume sufficient amounts of ethanol to achieve pharmacologically relevant blood ethanol concentrations (BEC) if they have not been genetically selected for their high ethanol preference or exposed to ethanol during early life (in utero or at adolescence) [START_REF] Alaux-Cantin | Alcohol intoxications during adolescence increase motivation for alcohol in adult rats and induce neuroadaptations in the nucleus accumbens[END_REF][START_REF] Mcbride | The alcohol-preferring (P) and high-alcohol-drinking (HAD) rats--animal models of alcoholism[END_REF]. Despite these challenges, the present article presents new evidence indicating that a rodent model of BD is now possible. Such models allow greater control over environmental parameters, and thereby enable systematic testing of those individual and environmental factors that may promote voluntary BD behavior. Ultimately, animal models of BD will address identification of neurobiological factors and how the speed of drinking may be the keystone factor in this behavior.

Although the number of publications on "BD in students" has increased since the beginning of the 1990's, the same increase among comparable papers describing animal models of BD began around 2010 (see Figure 1). Since 2008, more than 120 preclinical studies on BD have been published each year essentially on either rats or mice, but clearly, the animal literature has not kept pace with the burgeoning research on BD in students.

Relevant clinical criteria for developing animal models of BD

The specific behavioral pattern of BD is central to the development of an animal model of BD. Thus, the quantity, frequency, and duration parameters are crucial.

Concerning quantity, signs of intoxication (such as motor impairment, which is the most easily visible sign of intoxication in rodents) must be achieved with a sufficient amount of alcohol consumed in a short period of time. Even though intoxication is not part of the clinical definition, pharmacologically relevant BECs of approximately 1g/l need to be achieved, especially because rodents metabolize alcohol much more rapidly than humans (in rats, 3 times greater, or 300mg/kg/h and in mice, 5.5 times greater, or 550mg/kg/h). Figure 2 depicts BEC magnitude and time-course in humans, rats and mice after binge-like ethanol administration of about 0.5 to 0.75g/kg, and illustrates that rodents metabolize ethanol faster than humans. With respect to frequency and thus the history of the BD behavior in humans, this factor is very heterogeneous depending on the studies. For example, some studies investigated the BD behavior during the past month, the past 6 or 12 months or other durations before the recruitment. Furthermore, only few studies report dependence as an exclusion criterion in the BD studies, this should be clarified because BD and dependence are two different entities at the nosographical level. The BD behavior is more characterized by episodic drinking (intermittent consumption interspersed with longer or shorter periods of non-consumption) while dependence is more reflected by a daily consumption. Recently, in one study focusing on BD in humans [START_REF] Morris | Binge drinking differentially affects cortical and subcortical microstructure[END_REF] dependence was an exclusion criterion. In addition, in our previous study on BD in humans (Rolland et al., 2017), in the absence of DMS-5 diagnostic, a daily heavy consumption of alcohol, closely related to dependence diagnostic was also considered an exclusion criterion. This distinction is actually ambiguous since only very few studies on BD evaluated the dependence criterion and on the same vein, very few study studying dependence focused on BD patterns. Consideration of dependence in BD studies is important because it is also possible that a certain proportion of binged drinkers may be already dependent.

The frequency of BD is in fact important to consider, since different populations of binge drinkers can be distinguished according to this parameter, e.g., "frequent" or "infrequent" binge drinkers and animals can thus be used to model either the impact of very few or that of repeated BD episodes. The frequency of BD and individual history are important since chronic BD (i.e., for several months or years) is a risk factor for alcohol addiction in humans [START_REF] Kuntsche | Binge drinking: Health impact, prevalence, correlates and interventions[END_REF]. Moreover, it is frequently not clear whether participants in the clinical population recruited for BD studies met criteria for dependence (although this diagnosis has been removed from the DSM-5) (Rolland and Naassila, 2017). Finally, the duration, or the time period over which drinking occurs, is the most important parameter to identify BD because given equal quantities consumed, heavy drinking becomes BD as duration shortens.

Underage BD is a major health concern in several countries. Thus, using a developmental approach with animal models of BD could determine the mechanisms underlying long-term vulnerability for alcohol dependence. For example, several rodent studies have used forced and repeated (intermittent) ethanol administration during the adolescence period in rodents (the second month of life). This research has showed that increased risk for consuming alcohol in adulthood may involve reward deficit syndrome [START_REF] Alaux-Cantin | Alcohol intoxications during adolescence increase motivation for alcohol in adult rats and induce neuroadaptations in the nucleus accumbens[END_REF]. Interestingly, it has been shown that intermittent access to voluntary ethanol drinking facilitates excessive drinking after rats are subsequently exposed to chronic intermittent ethanol vapor [START_REF] Kimbrough | Intermittent Access to Ethanol Drinking Facilitates the Transition to Excessive Drinking After Chronic Intermittent Ethanol Vapor Exposure[END_REF]. Thus, the frequency and the pattern of alcohol drinking may play a crucial role in vulnerability to dependence.

Clinical studies have shown that BD is associated with impairment in executive function, such as inhibitory control and decision-making (Lannoy et al., 2017b). BD is also associated with alterations in grey and white matter that are correlated with cognitive impairments [START_REF] Hermens | Pathways to alcohol-induced brain impairment in young people: a review[END_REF]. Interestingly some results have revealed that BD is associated with impaired performance on cognitive tasks in women more than men [START_REF] Townshend | Binge drinking, cognitive performance and mood in a population of young social drinkers[END_REF]. Thus, both brain alterations and cognitive impairments (e.g. in prefrontal executive functions or memory) must be investigated in animal models of BD, and differences linked to gender, as well as interactions between BD and gender differences in brain maturation, should be explored. At the somatic level, some studies suggest that BD, independently of daily alcohol intake, can lead to more severe forms of alcoholic liver disease in younger populations [START_REF] Ventura-Cots | Binge drinking as a risk factor for advanced alcoholic liver disease[END_REF].

Current preclinical models of BD

Preclinical research on alcohol also suffers from this lack of a clear definition of BD behavior. Animal models attempt to parallel the human condition, and in fact, the NIAAA definition of BD in humans has been used in animal studies (Crabbe et al., 2011a).

Published studies on BD using the nonhuman primate model are scarce. Adolescent nonhuman primate model has been used to investigate the impact of BD on hippocampal neurogenesis using oral ethanol self-administration induced with a procedure in which the concentration of ethanol in a palatable solution (Tang) was gradually increased (1 to 6%) over a series of daily limited-access sessions (1-h) for 11 months (Taffe et al., 2010.). In this model nonhuman primates consumed an average of 1.74g/kg of alcohol during the 1-h daily sessions and they reached BEC of 50 to 300 mg/dl when measured 30min after the beginning of the session. Another study in primate model on the transgenerational effects of BD used a forced administration of increasing doses (0.75 to 1.5 g/kg) of ethanol (mean BEC at 3-h post administration of 140.3±31.3 mg/dl) via nasogastric tube twice weekly for 6 months (VandeVoort et al., 2015).

In the present review we have chosen to focus on rodent models of BD for which we have the most data compared to other species.

Current animal models of BD can be divided into two large categories depending on the mode of administration: forced/passive administration of high alcohol doses or voluntary/active alcohol consumption.

Forced administration of high doses of alcohol

In this first category, animals receive high doses of alcohol solution leading to rapid elevation of BEC and clear signs of intoxication. These procedures are easy to perform and do not require the acquisition of specific equipment; however, as we will discuss later, the passive administration of a drug does not produce the same neurobiological and behavioral modifications as voluntary consumption, and therefore, may be of limited use, depending on the scientific question.

Gavage:

This simple procedure consists of the insertion of a guide cannula into the esophagus of the rodent and the injection of an ethanol solution (usually a 10 or 20 % ethanol solution in tap water). Through absorption in the digestive tract, BEC will increase rapidly, reflecting the pharmacokinetics observed in humans [START_REF] Yang | Neurons in the deep layers of superior colliculus are a requisite component of the neuronal network for seizures during ethanol withdrawal[END_REF]. With a single administration, BECs can reach more than 500 mg/dL, sustained for several hours, after a gavage of 7g/kg (32% ethanol solution v/v) [START_REF] Carson | Development and characterization of a binge drinking model in mice for evaluation of the immunological effects of ethanol[END_REF]. In mice, more moderate and ecologically valid BECs generally are produced (e.g., 120 mg/dL for 30 minutes after a gavage of 1.12g/kg of a 10% ethanol solution) [START_REF] Chen | Intoxication by intraperitoneal injection or oral gavage equally potentiates postburn organ damage and inflammation[END_REF]. Major differences exist between mice and rats using the gavage procedure. For example, 300 and 100 mg/dL are achieved after a gavage of 3.8 g/kg of a 21% ethanol solution in mice and rats, respectively [START_REF] Livy | Blood ethanol concentration profiles: a comparison between rats and mice[END_REF]. Mice show a faster rise and higher peak BEC and elimination rates while rats show more gradual pharmacokinetics profiles and retain ethanol in their bloodstream for longer periods (Figure 2). The gavage procedure can be performed acutely [START_REF] Jin | Diminished neutrophil extracellular trap (NET) formation is a novel innate immune deficiency induced by acute ethanol exposure in polymicrobial sepsis, which can be rescued by CXCL1[END_REF][START_REF] Liu | Ethanol Induces Platelet Apoptosis[END_REF] or in subchronic [START_REF] Thompson | Use of a crossed high alcohol preferring (cHAP) mouse model with the NIAAA-model of chronic-binge ethanol intake to study liver injury[END_REF] and chronic procedures [START_REF] Hansen | Taurine restores the exploratory behavior following alcohol withdrawal and decreases BDNF mRNA expression in the frontal cortex of chronic alcohol-treated rats[END_REF], but with the limitation that the direct administration of large volume of an ethanol solution in the stomach will induce direct damage to the organ [START_REF] Kawashima | Alcohol injury to gastric mucosa in mice and its potentiation by stress[END_REF]Jerzy Glass, 1975, Chen et al., 2013). In addition, the repeatability of the results are dependent of the gastric contents at the time of administration, and it has been well described that ethanol administration by gavage will have different consequences (absorption, pharmacokinetic, BECs reached, behavioral consequences) when the stomach is empty or not [START_REF] Roine | Effect of concentration of ingested ethanol on blood alcohol levels[END_REF]. Thus, the gavage procedure has limited utility to investigate BD (Livy et al., 2003, Walker and[START_REF] Walker | Age-related differences in the blood alcohol levels of Wistar rats[END_REF]. Although a large variety of studies on liver injury has been performed using this paradigm [START_REF] Thompson | Use of a crossed high alcohol preferring (cHAP) mouse model with the NIAAA-model of chronic-binge ethanol intake to study liver injury[END_REF][START_REF] Wang | Inflammation is independent of steatosis in a murine model of steatohepatitis[END_REF][START_REF] Yao | Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation[END_REF], fewer studies have used it in behavioral neuroscience [START_REF] Griffin | Intensity and duration of chronic ethanol exposure is critical for subsequent escalation of voluntary ethanol drinking in mice[END_REF][START_REF] Anton | Oleoylethanolamide prevents neuroimmune HMGB1/TLR4/NF-kB danger signaling in rat frontal cortex and depressive-like behavior induced by ethanol binge administration[END_REF][START_REF] Karelina | Binge ethanol in adulthood exacerbates negative outcomes following juvenile traumatic brain injury[END_REF]. Nevertheless, some utility of the gavage procedure has been recognized because a combination of the Lieber-DeCarli regimen (forced consumption of alcohol through the sole source of nutrients) and the gavage procedure have been recently identified as the NIAAA model of chronic and binge ethanol feeding (Bertola et al., 2013a, Bertola et al., 2013b).

Intra-peritoneal injections:

This procedure is easier to implement than the gavage, partially because intraperitoneal injections may induce less stress than the gavage procedure. There are also large differences between rats and mice using the intra-peritoneal route, and like gavage, the peak BECs are lower in rats than in mice [START_REF] Livy | Blood ethanol concentration profiles: a comparison between rats and mice[END_REF]. Most of the time, these procedures are used acutely and sometimes sub-chronically with a specific schedule of administration, and single or multiple discrete injections of alcohol have been used extensively to study the effect of acute intoxication in rodents. Moderate to high doses (i.e., around 2 to 3 g of pure ethanol/kg of body weight for the mice and 1.5 to 3g/kg for the rats) are typically tested in order to induce intoxication without sedative/hypnotic effects (measured with the loss of the righting reflex). In the Naassila laboratory, we have used a protocol of 2 consecutive injections of ethanol administered 9 hrs apart to mimic a double binge-like episode and to determine if only two BD episodes are sufficient to adversely affect memory and synaptic plasticity. Indeed, using this procedure in adolescent rats, Silvestre de Ferron and colleagues (Silvestre de [START_REF] De Ferron | Two Binges of Ethanol a Day Keep the Memory Away in Adolescent Rats: Key Role for GLUN2B Subunit[END_REF] demonstrated that only 2 intoxications induced by injections of 3g/kg of ethanol, lead to a complete deletion of the specific form of synaptic plasticity called long-term depression. The abolition of this specific form of synaptic plasticity may be responsible for a perturbation of memory processes assessed in the novel object recognition test. Several variants of this procedure can be found in the literature. For example, Pascual et al. [START_REF] Pascual | Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats[END_REF]) developed a relevant protocol in which rats received a total of 8 intraperitoneal injections following a schedule of 1 daily injection for 2 days in a row, then 2 days off, and so on, until rats received the 8 injections of a 25% ethanol solution at a dose of 3 g/kg. This protocol mimics BD-like ethanol exposure during the adolescent period (postnatal day 25 to 38) as observed in humans and also to introduce withdrawal-like period with the 2-day off intervals. This protocol has also been successfully used in mice with identical injections from postnatal 30 to 43 (Pascual et al., 2017b) to study inflammatory mechanisms following binge-like exposure. [START_REF] Pascual | Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats[END_REF] found that this pattern of alcohol injections leads to behavioral alterations (evaluated in a conditioned discrimination learning task) and is associated with brain damage linked to apoptosis and neuroinflammation. Numerous studies have now been published using this procedure [START_REF] Pascual | Changes in histone acetylation in the prefrontal cortex of ethanolexposed adolescent rats are associated with ethanol-induced place conditioning[END_REF][START_REF] Alaux-Cantin | Alcohol intoxications during adolescence increase motivation for alcohol in adult rats and induce neuroadaptations in the nucleus accumbens[END_REF][START_REF] Montesinos | Involvement of TLR4 in the long-term epigenetic changes, rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence[END_REF].

One of the limitations of these intra-peritoneal injections of alcohol procedures is the local pain and irritation induced by the injection of a large volume of a solution of ethanol (10 to 20% usually). Repetition of injections may result in an increase of stress and local inflammation associated with damages to different organs (D'Souza [START_REF] El-Guindy | Laboratory models available to study alcohol-induced organ damage and immune variations: choosing the appropriate model[END_REF].

Inhalation of alcohol vapors:

The alcohol vapor inhalation model was developed to induce physical signs of alcohol dependence by arranging long intoxication periods for several weeks or months, with target BECs of 175-250 mg/dL (Le [START_REF] Bourhis | Alcohol intoxication via the lung in rats][END_REF][START_REF] Bourhis | Pattern of alcohol administration and physical dependence[END_REF][START_REF] Schulteis | Decreased brain reward produced by ethanol withdrawal[END_REF][START_REF] Simon-O'brien | The histone deacetylase inhibitor sodium butyrate decreases excessive ethanol intake in dependent animals[END_REF]. More recently, and to better model the observed pattern of alcohol consumption in humans with alcohol addiction, intermittent exposure has been introduced every day (usually 14h on/10h off per day). These durations of intoxication seem too long to be associated with binge-like intoxication. To our knowledge, short periods of exposure to ethanol vapors have not been used to mimic BD.

Forced drinking:

For this procedure, rats or mice are kept usually in their home cage and instead of having water delivered ad libitum, alcohol is the sole source of fluid. Thus, rodents have to drink the alcohol solution to survive. The scheduled high alcohol consumption is another paradigm in which availability of water is restricted; in order to survive, rats must drink more ethanol as a function of reduced access to water [START_REF] Cronise | Scheduled access to ethanol results in motor impairment and tolerance in female C57BL/6J mice[END_REF]. Another forced consumption of alcohol can be obtained using a liquid diet procedure developed by Lieber and DeCarli [START_REF] Lieber | Alcoholic liver injury: experimental models in rats and baboons[END_REF].

Rodents have access to a bottle containing ethanol (4 to 9 % w/v) and all the necessary nutrients for several weeks. Intoxicating levels are easily reached (100 mg/dL in Wistar rats, [START_REF] Weiss | Ethanol self-administration restores withdrawal-associated deficiencies in accumbal dopamine and 5-hydroxytryptamine release in dependent rats[END_REF] and until 180 mg/dL in C57 mice (Bertola et al., 2013a). Despite the somewhat forced nature of consumption, bouts are titrated over a 24-h period and to our knowledge no study has demonstrated that rats could display BD-like behavior using this procedure when ethanol solution is available 24-h a day and not only during a restricted period of time of the day. However, using these procedures, rats can consume more than 7g/kg ethanol/day, thereby exceeding their daily ethanol metabolism capacity [START_REF] Aufrere | Ethanol intake after chronic intoxication by inhalation of ethanol vapour in rats: behavioural dependence[END_REF]. It is of note that forced drinking may be linked to a reduction in daily total fluid intake depending on the strain [START_REF] Azarov | Early ethanol and water intake: choice mechanism and total fluid regulation operate in parallel in male alcohol preferring (P) and both Wistar and Sprague Dawley rats[END_REF] which may have moderate dehydration consequence [START_REF] Blank | Suppression of natural killer cell activity by ethanol consumption and food restriction[END_REF] leading to stress and thus altering behavioral and physiological parameters.

Summary of the studies on ethanol forced administration

All of the forced-ethanol administration procedures described above have the ability to induce alcohol intoxication and allow the researchers to observe direct consequences of binge-like intoxication. In addition, they are simple to set up in all laboratories, with no need for expensive equipment, and can rapidly generate experimental animals displaying high level of ethanol intake. Following such procedures, several types of experiments can be performed, such as genetic testing [START_REF] Pascual | Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats[END_REF], behavioral studies [START_REF] Montesinos | Involvement of TLR4 in the long-term epigenetic changes, rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence[END_REF], neuroinflammation investigation [START_REF] Pascual | Cytokines and chemokines as biomarkers of ethanol-induced neuroinflammation and anxiety-related behavior: role of TLR4 and TLR2[END_REF], in addition to study of the microbiota [START_REF] Chen | Microbiota Protects Mice Against Acute Alcohol-Induced Liver Injury[END_REF] and liver injury (Wilkin et al., 2016). However, these paradigms are limited as models of human BD because consumption cannot be viewed as voluntary. Therefore, it is difficult, or even impossible, to evaluate some important parameters of drug consumption such as the motivation to consume and the seeking for the drug in a non-operant paradigm. Finally, even though the gavage procedure (i.e., via the oral route) may appear to have greater face validity compared with the intra-peritoneal route, it should be considered that, at least in rats, its ability to mimic BD is limited since the achieved peak BECs and alcohol elimination rates are low.

Voluntary alcohol consumption

In contrast to forced alcohol administration, voluntary alcohol consumption usually does not result in sufficient levels of BEC to induce any behavioral signs of intoxication, at least in outbred animals that have not been genetically selected for their ethanol preference. Recently, several protocols, mostly in mice and sometimes in rats, have been developed to induce high levels of BEC. One long-standing method that has been used successfully to enhance voluntary alcohol intake is the scheduling of alcohol availability (Le [START_REF] Magnen | Study of some factors associated with modifications of spontaneous ingestion of ethyl alcohol by the rat[END_REF]. When animals have unlimited access to alcohol, the consumption bouts are usually titrated over a 24-h period.

Thus, even though significant pharmacologically relevant BEC may be obtained at some points during the day, animals do not display visible signs of motor impairment.

Therefore, other models have been developed to achieve elevated BECs in different periods of time.

20% alcohol intake in the two-bottle choice intermittent access model:

In 1973, Roy Wise described a protocol of voluntary alcohol consumption using a 2bottle-choice paradigm in which rats have access to one bottle of tap water and one of alcohol (20% ethanol solution) every other day [START_REF] Wise | Voluntary ethanol intake in rats following exposure to ethanol on various schedules[END_REF]. Using this protocol, rats demonstrate increased consumption, leading to alcohol intake of more than 5g/kg per day when alcohol is available. Interestingly, a recent study revealed that multiple episodes of excessive alcohol consumption using this model (3 days per week for 7 weeks) leads to somatic damage measured as liver metabolism dysfunction and inflammation [START_REF] Wegner | Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice[END_REF]. This procedure found a new revival in the beginning of the 2000s with several publications [START_REF] Simms | Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats[END_REF], Carnicella et al., 2009[START_REF] Hopf | Motivation for alcohol becomes resistant to quinine adulteration after 3 to 4 months of intermittent alcohol self-administration[END_REF][START_REF] Simon-O'brien | The histone deacetylase inhibitor sodium butyrate decreases excessive ethanol intake in dependent animals[END_REF], Spoelder et al., 2017a, Spoelder et al., 2017b). A major advantage of this procedure is that it can be performed with inbred animals, such as Fischer rats, [START_REF] Mill | Fischer rats consume 20% ethanol in a long-term intermittent-access two-bottle-choice paradigm[END_REF] and C57BL/6J mice [START_REF] Hwa | Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol[END_REF] and alcohol-preferring lines including the Sardinian P rats [START_REF] Sabino | Pharmacological characterization of the 20% alcohol intermittent access model in Sardinian alcohol-preferring rats: a model of binge-like drinking[END_REF], as well as with outbred rats such as Long Evans [START_REF] Carnicella | GDNF is a fastacting potent inhibitor of alcohol consumption and relapse[END_REF][START_REF] Hamida | The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors[END_REF][START_REF] Meyer | Stress increases voluntary alcohol intake, but does not alter established drinking habits in a rat model of posttraumatic stress disorder[END_REF], Wistar [START_REF] Wise | Voluntary ethanol intake in rats following exposure to ethanol on various schedules[END_REF][START_REF] Simms | Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats[END_REF][START_REF] Hopf | Motivation for alcohol becomes resistant to quinine adulteration after 3 to 4 months of intermittent alcohol self-administration[END_REF][START_REF] Cippitelli | Pharmacological blockade of corticotropin-releasing hormone receptor 1 (CRH1R) reduces voluntary consumption of high alcohol concentrations in non-dependent Wistar rats[END_REF] and Sprague-Dawley rats [START_REF] Bito-Onon | Varenicline, a partial agonist at neuronal nicotinic acetylcholine receptors, reduces nicotineinduced increases in 20% ethanol operant self-administration in Sprague-Dawley rats[END_REF]. This procedure is normally used to study long-term alcohol consumption over a 24-h period; however, animals will consume a large part of their daily total intake only during the first hours of presentation of the alcohol bottle.

Depending on the laboratory, the duration of this binge-like consumption is measured during the first 30 minutes [START_REF] Simms | Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats[END_REF], Carnicella et al., 2009[START_REF] Hamida | The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors[END_REF], the first hour [START_REF] Sabino | Pharmacological characterization of the 20% alcohol intermittent access model in Sardinian alcohol-preferring rats: a model of binge-like drinking[END_REF] or the first two hours [START_REF] Hwa | Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol[END_REF]. The total amount of ethanol consumed during the first 30 minutes represents at least 20% of the total amount consumed in 24-h and leads to averaged BECs of 80 mg/dL with some animals showing BECs above 100 mg/dL [START_REF] Carnicella | Excessive alcohol consumption is blocked by glial cell line-derived neurotrophic factor[END_REF]. Similar results were obtained with the Sardinian P rats with an average BEC around 80 mg/dL but with only very few animals reaching more than 100 mg/dL after 1-h of consumption [START_REF] Sabino | Pharmacological characterization of the 20% alcohol intermittent access model in Sardinian alcohol-preferring rats: a model of binge-like drinking[END_REF]. In their elegant study, Simms et al., compared different strains of rats and showed that in 30-min, the Long Evans rats exhibited higher BECs and higher interindividual variability than Wistar and P rats [START_REF] Simms | Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats[END_REF]. Thus, their results suggest that the Long Evans may be most suitable for BD studies.

The predictive validity of this BD model has also been demonstrated. Indeed naltrexone decreased the total amount of alcohol consumed during the first 30 min of the drinking episode from 1.1 to 0.6 g/kg/30 min [START_REF] Simms | Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats[END_REF]. Moreover, varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist already used in smoking cessation leads to a decrease in ethanol consumed in this model from 1.4 to 0.4 g/kg/30 min [START_REF] Steensland | Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking[END_REF].

In this 20% ethanol intermittent access model, the escalation of ethanol intake may be associated with sensitization since it develops in a few weeks and then reaches a plateau, and once established, can last even after 40 days of ethanol intake discontinuation [START_REF] Simms | Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats[END_REF]. This point is very interesting in the context of BD behavior since it reflects the fact that episodic ethanol intake may profoundly impact brain functioning and therefore, behavior.

Two bottle choice drinking-in-the-dark model:

Currently, the most frequently used model to mimic BD is the drinking-in-the-dark (DID) procedure, in which animals drink large amounts of ethanol during the first hours of the dark cycle, either immediately upon lights out (for rats) or after 3 or 4 hours (for mice) and display BEC in excess of 0.8g/L [START_REF] Thiele | Drinking in the Dark" (DID): a simple mouse model of binge-like alcohol intake[END_REF]. There are several variations on the DID model, depending on the laboratory performing the experiment: some use a one-bottle paradigm [START_REF] Rhodes | Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice[END_REF][START_REF] Crabbe | A line of mice selected for high blood ethanol concentrations shows drinking in the dark to intoxication[END_REF][START_REF] Neasta | Role for mammalian target of rapamycin complex 1 signaling in neuroadaptations underlying alcohol-related disorders[END_REF], others a 2-bottle paradigm [START_REF] Kaur | Ghrelin receptor antagonism decreases alcohol consumption and activation of perioculomotor urocortin-containing neurons[END_REF]Ryabinin, 2010, Burnham and[START_REF] Burnham | Voluntary Binge-like Ethanol Consumption Sitespecifically Increases c-Fos Immunoexpression in Male C57BL6/J Mice[END_REF], and even some others a 4-bottle choice paradigm [START_REF] Colombo | Binge drinking in alcohol-preferring sP rats at the end of the nocturnal period[END_REF]. Similarly, the time of the beginning of the drinking sessions within the dark phase can vary from 1 to 4 hours and the duration of the drinking episode can be of 2 to 4 hours [START_REF] Rhodes | Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice[END_REF]. The major caveat of this procedure is that it seems to produce intoxicating levels only in inbred animals known to show spontaneous alcohol preference. Indeed, the same group of researchers demonstrated that this DID model leads to intoxication (BECs > 80 mg/dL) only in C57BL/6J mice when compared with 11 other strains of mice [START_REF] Rhodes | Mouse inbred strain differences in ethanol drinking to intoxication[END_REF].

In a recent study using the Sardinian P rats, [START_REF] Colombo | Binge drinking in alcohol-preferring sP rats at the end of the nocturnal period[END_REF] demonstrated that the start time of the drinking phase plays a role in the amount of alcohol consumed in a 4-bottle choice paradigm. Indeed if the session of drinking starts 1 hour prior to the end of the dark phase of the day, rats will consume 3 times more alcohol than if the session starts at the beginning of the dark phase. The rats consumed up to 2g/kg of pure ethanol in 1 hour, thereby leading to intoxication (BECs around 100 mg/dL) and symptoms of such intoxication revealed by rotarod impairment [START_REF] Colombo | Binge drinking in alcohol-preferring sP rats at the end of the nocturnal period[END_REF].

A multiple scheduled access (MSA) has been added to the DID procedure in which animals experience between two and four-hour access periods across the 12-h dark cycle with each access period separated by 2 or more hours. However, this DID-MSA procedure has been used mainly in alcohol-preferring rats [START_REF] Bell | Gene expression changes in the nucleus accumbens of alcohol-preferring rats following chronic ethanol consumption[END_REF][START_REF] Mcbride | Changes in gene expression in regions of the extended amygdala of alcoholpreferring rats after binge-like alcohol drinking[END_REF] and not outbred animals, and thus, it does not allow for generalization to highly heterogeneous populations. One other caveat of this procedure is that it usually takes place within a week and thus does not allow the establishment of a robust chronic consumption of elevated levels of alcohol. One study using the DID procedure aimed to overcome this problem by providing access to the bottle for 6 weeks [START_REF] Wilcox | Repeated binge-like ethanol drinking alters ethanol drinking patterns and depresses striatal GABAergic transmission[END_REF]. The authors found that the consumption was stable over this extended period of DID. In addition, they found that this prolonged chronic bingelike consumption of alcohol led to neurobiological alterations, such as depressed GABAergic transmission and modifications of neuron excitability (frequency of discharge), with a shift from the dorsomedial striatum to an activation of the dorsolateral part of the striatum after a prolonged experience of DID in response to an acute administration of alcohol [START_REF] Wilcox | Repeated binge-like ethanol drinking alters ethanol drinking patterns and depresses striatal GABAergic transmission[END_REF].

The predictive validity of the DID model was confirmed using naltrexone, one clinically recognized treatment for alcohol addiction, and also using the dopamine reuptake blocker GBR 12909. Both drugs were able to dose-dependently decrease ethanol consumption in this model [START_REF] Kamdar | Acute effects of naltrexone and GBR 12909 on ethanol drinking-in-the-dark in C57BL/6J mice[END_REF].

The DID procedure also has been used to create a selected mouse line, akin to the alcohol-preferring P rat. Crabbe and his colleagues selected over 11 generations of mice, called the High Drinking-in-the-Dark mice (HDID) exhibiting the highest levels of ethanol consumption in this procedure [START_REF] Crabbe | A line of mice selected for high blood ethanol concentrations shows drinking in the dark to intoxication[END_REF], Crabbe et al., 2011b). This selection produced average BECs above 100 mg/dL with a maximum of 250 mg/dL in 2 hours of consumption. The predictive validity of the HDID model was tested using 3 different actual drugs used to treat alcohol-dependent patients, namely naltrexone, baclofen and acamprosate. Surprisingly, the authors found that only acamprosate, and with a high dose, was able to reduce ethanol consumption in the HDID mice, but without significantly reducing the BECs, when consumption was evaluated for 4 hours [START_REF] Crabbe | High Drinking in the Dark (HDID) mice are sensitive to the effects of some clinically relevant drugs to reduce binge-like drinking[END_REF]. When evaluated in the 2 first hours, baclofen was able to reduce both alcohol consumption and subsequent BECs, but non-selectively, as it also reduced water intake [START_REF] Crabbe | High Drinking in the Dark (HDID) mice are sensitive to the effects of some clinically relevant drugs to reduce binge-like drinking[END_REF]. Because naltrexone was not effective in the HDID model, and relatively high doses of drugs were required to reduce consumption, the HDID model lacks sufficient predictive validity, particularly because naltrexone is known for its efficacy in reducing high level of alcohol intake in patients who relapse (Jonas et al. 2014).

Operant binge drinking:

Voluntary ethanol consumption in the 2-bottle choice paradigm can lead to intoxication, but otherwise, provides little information concerning complex alcoholassociated behaviors. Conversely, operant self-administration of alcohol has been widely used to obtain such type of information (motivation, seeking, relapse after extinction…); however, even with the combination of a 20% intermittent access protocol to induce alcohol palatability in outbred rats, the total amount of alcohol consumed barely reached the levels of intoxication in periods of 30 minutes [START_REF] Jeanblanc | Endogenous BDNF in the dorsolateral striatum gates alcohol drinking[END_REF][START_REF] Carnicella | Regulation of operant oral ethanol selfadministration: a dose-response curve study in rats[END_REF][START_REF] Carnicella | Intermittent ethanol access schedule in rats as a preclinical model of alcohol abuse[END_REF][START_REF] Jeanblanc | The Class I-Specific HDAC Inhibitor MS-275 Decreases Motivation to Consume Alcohol and Relapse in Heavy Drinking Rats[END_REF][START_REF] Darcq | The Neurotrophic Factor Receptor p75 in the Rat Dorsolateral Striatum Drives Excessive Alcohol Drinking[END_REF].

Gilpin and colleagues have developed an operant model of binge-like alcohol selfadministration in adolescent Wistar rats [START_REF] Gilpin | Adolescent binge drinking leads to changes in alcohol drinking, anxiety, and amygdalar corticotropin releasing factor cells in adulthood in male rats[END_REF]. By adding a mixed solution of 3% glucose/0.125% saccharin to a 10% ethanol solution (w/v), the authors observed intakes exceeding 1 g/kg/30 min leading to BECs around 80 mg/dL. Moreover, the rats exhibiting high levels of ethanol self-administration during adolescence showed reduced anxiety at adulthood. We caution that the use of any type of sucrose should be avoided in animal models of excessive alcohol intake [START_REF] Carnicella | Intermittent ethanol access schedule in rats as a preclinical model of alcohol abuse[END_REF]. Sweeteners may reduce blood ethanol levels for a particular volume of ethanol consumed [START_REF] Roberts | Operant self-administration of sweetened versus unsweetened ethanol: effects on blood alcohol levels[END_REF] and saccharin can alter the dynamics of cue-ethanol memory reconsolidation (Puaud et al., 2018). Sucrose-sweetened ethanol and sucrose fading are frequently used in animal models of operant oral self-administration, but researchers should be wary of possible interactions with neurobiological and behavioral outcomes [START_REF] Carnicella | Intermittent ethanol access schedule in rats as a preclinical model of alcohol abuse[END_REF].

Recently, we demonstrated that a daily, very short session of operant selfadministration can facilitate the acquisition of BD behavior in rats [START_REF] Lebourgeois | Effect of N-acetylcysteine on motivation, seeking and relapse to ethanol selfadministration[END_REF] (Jeanblanc et al., 2018). After several weeks of training under a fixed ratio 3 (3 lever presses to get 0.1ml of 20% ethanol) during 1 hour, and thereafter reduced to 30 minutes (high drinkers), we further reduced the duration of the daily sessions to only 15 minutes (binge drinkers). We observed an increase in ethanol consumption (Figure 3) reaching about 1.2 g/kg and rats displayed clear signs of intoxication (i.e. sedation and ataxia: loss of locomotor coordination). The pattern of alcohol deliveries and the number of lever presses observed in our models are presented in Figure 4.

The pattern of alcohol deliveries of the BD rats clearly shows that these rats display a high rate of ethanol consumption.

After several weeks of chronic daily BD sessions, we also observed signs of withdrawal (e.g., aggressive behavior and vocalizations) and an increased motivation to consume alcohol even for a highly concentrated solution (30% ethanol). Finally, we also observed somatic damage in BD rats with these animals displaying typical signs of hepatic steatosis (Figure 5). In this model, we demonstrate that rats can consume very quickly, as is observed in human BD and as would be expected in a relevant animal model of BD.

Criteria for an animal model of BD

Here we propose seven criteria (see Table 1) that we believe need to be met for the behavior of BD in an animal model to correspond to that in humans and therefore, to serve as a valid model of human BD (Figure 6).

The first criterion is voluntary ethanol intake by oral ingestion. Numerous studies have shown that forced (passive) administration of addictive substances is not appropriate to decipher etiological factors involved in behavior compared with active administration. In addition, it is generally accepted that paradigms in which drug administration is contingent on an instrumental response (self-administration) are a better model for human addiction [START_REF] Jacobs | Neuroadaptive effects of active versus passive drug administration in addiction research[END_REF]. Active self-administration behavior affects the nature and direction of drug-induced neuroplasticity [START_REF] Jacobs | Neuroadaptive effects of active versus passive drug administration in addiction research[END_REF] and may also involve an expectancy phenomenon (i.e., expecting to receive the drug is known to potentiate brain activation in humans) [START_REF] Volkow | Expectation enhances the regional brain metabolic and the reinforcing effects of stimulants in cocaine abusers[END_REF]. For example, studies on cocaine have demonstrated that active self-administration has a greater effect on dopamine release compared with yoked or non-contingent delivery [START_REF] Lecca | Differential neurochemical and behavioral adaptation to cocaine after response contingent and noncontingent exposure in the rat[END_REF][START_REF] Wiskerke | Response contingency directs long-term cocaine-induced neuroplasticity in prefrontal and striatal dopamine terminals[END_REF]. Concerning ethanol, [START_REF] Gilpin | Adolescent binge drinking leads to changes in alcohol drinking, anxiety, and amygdalar corticotropin releasing factor cells in adulthood in male rats[END_REF] have shown that voluntary (but not involuntary) BD during adolescence increases baseline drinking in adulthood in Wistar rats. Furthermore, oral ingestion clearly should be preferred to achieve face validity of the model and to mimic human drinking. In addition, food deprivation and sugar adulteration of the ethanol solution should be avoided because these manipulations may have an impact on motivation of the animal to consume ethanol and ethanol metabolism [START_REF] Roberts | Operant self-administration of sweetened versus unsweetened ethanol: effects on blood alcohol levels[END_REF], and can alter the dynamics of cueethanol memory reconsolidation (Puaud et al., 2018). Finally, as indicated above, the operant paradigm is preferred because it allows the study of other criteria that are central to the transition to dependence and some of its features, such as loss of control, compulsivity, high seeking (craving), motivation, loss of cognitive flexibility, behavioral sensitization and habit learning. This latter point is especially important since BD may be involved in the vulnerability to ethanol addiction [START_REF] Gowin | Vulnerability for Alcohol Use Disorder and Rate of Alcohol Consumption[END_REF]. Thus, it is crucial that addictive behaviors can be analyzed in the animal model of BD because, as in humans, it is possible that some binge drinkers may be categorized as dependent.

Second, sufficient levels of ethanol have to be consumed to achieve significant BEC and thus to induce visible signs of intoxication. Humans drinking ethanol in a BD pattern are seeking drunkenness and in some cases to ethylic coma. The 0.08g/dl threshold in the NIAAA definition of BD may also serve as a useful BEC threshold in animal models of BD since signs of intoxications are also visible in animals (even in outbred animals) that reach this BEC threshold after voluntary ethanol intake (Jeanblanc et al., 2018). A recent study in humans indicates that thresholds in alcohol quantity (such as ≤56g (moderate drinkers), 70g+ (binge drinkers) and 140g+ (extreme binge drinkers) of ethanol per occasion are an important factor to consider since some cognitive abilities (e.g., acquisition of new verbal information) may be particularly affected, particularly in extreme binge drinkers [START_REF] Nguyen-Louie | Learning and Memory in Adolescent Moderate, Binge, and Extreme-Binge Drinkers[END_REF].

Third, the drinking episode should be short and thus, animals should drink fast. We described here a study indicating that outbred rats can display signs of intoxication 15 minutes after the beginning of the drinking episode. Paradigms with drinking episodes of several hours may not demonstrate sufficient face validity because rodents metabolize ethanol more quickly compared with humans.

Fourth, drinking episodes should be both intermittent and repeated several days a week to model BD patterns as it may be observed in adolescent populations [START_REF] Courtney | Binge drinking in young adults: Data, definitions, and determinants[END_REF]. However, the pattern of BD may not correspond to everyday drinking as it is seen in ethanol dependent population (Rolland and Naassila, 2017). Thus, the fifth criterion specifies a few days without ethanol access should be included in the BD paradigm. Intermittent exposure to ethanol drinking is an important factor in the vulnerability to develop ethanol dependence [START_REF] Kimbrough | Intermittent Access to Ethanol Drinking Facilitates the Transition to Excessive Drinking After Chronic Intermittent Ethanol Vapor Exposure[END_REF] and produce brain damage [START_REF] Reynolds | Ethanol withdrawal is required to produce persisting N-methyl-D-aspartate receptor-dependent hippocampal cytotoxicity during chronic intermittent ethanol exposure[END_REF]. The duration of episodic alcohol exposures should be from subchronic to chronic in the context of BD studies.

Since several studies in humans and animal models of BD have demonstrated both brain damage/cognitive deficits (Townshend andDuka, 2005, McQueeny et al., 2009) [START_REF] Squeglia | The effect of alcohol use on human adolescent brain structures and systems[END_REF][START_REF] Smith | Apoptotic cell death and temporal expression of apoptotic proteins Bcl-2 and Bax in the hippocampus, following binge ethanol in the neonatal rat model[END_REF][START_REF] Salas-Gomez | Binge Drinking in Young University Students Is Associated with Alterations in Executive Functions Related to Their Starting Age[END_REF][START_REF] Cantacorps | Maternal alcohol binge drinking induces persistent neuroinflammation associated with myelin damage and behavioural dysfunctions in offspring mice[END_REF][START_REF] Cohen-Gilbert | College Binge Drinking Associated with Decreased Frontal Activation to Negative Emotional Distractors during Inhibitory Control[END_REF][START_REF] Molnar | Behavioral and Brain Activity Indices of Cognitive Control Deficits in Binge Drinkers[END_REF] and somatic damage [START_REF] Llerena | Binge drinking: Burden of liver disease and beyond[END_REF][START_REF] Mostofsky | Risk of myocardial infarction immediately after alcohol consumption[END_REF], the sixth criterion is that an animal model of BD should display this kind of damage [START_REF] Ventura-Cots | Binge drinking as a risk factor for advanced alcoholic liver disease[END_REF][START_REF] Wegner | Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice[END_REF]. Studies in both humans and animals have shown that BD adversely affects both white and grey matter and that the neuro-inflammatory processes play an important role in ethanol-induced neurotoxicity [START_REF] Jacobus | Neurotoxic effects of alcohol in adolescence[END_REF][START_REF] Crews | Adolescent Alcohol Exposure Persistently Impacts Adult Neurobiology and Behavior[END_REF], Pascual et al., 2017a[START_REF] Saba | Imaging the neuroimmune response to alcohol exposure in adolescent baboons: a TSPO PET study using (18) F-DPA-714[END_REF].

Finally, as stated above BD is a complex behavior and binge drinkers represent a heterogeneous population [START_REF] Nguyen-Louie | Learning and Memory in Adolescent Moderate, Binge, and Extreme-Binge Drinkers[END_REF], Gierski et al., 2017). Thus, it is important to be able to study such an interindividual variability in an animal model of BD, which constitutes our seventh criterion.

Concluding remarks and need for future research

The choice of the animal species and the procedure of alcohol administration are crucial to create an appropriate animal model of BD for addressing both neurobiological and behavioral aspects. All the procedures discussed here have their advantages and their limitations. Each project and each scientific question need to be a priori challenged with the different models to ensure the most adequate choice.

Animal models using forced/passive ethanol administration are largely used to explore brain and somatic consequences of BD. The other models using voluntary intake may display better face validity since they allow investigations of both the impact of the high speed of drinking and the motivational aspect of BD that may be useful to investigate the transition to alcohol dependence. Therefore, animal models of BD using voluntary intake should be preferred for future integrative and/or translational research especially because recent data indicate that this procedure can lead to very fast alcohol intake and induce both cognitive and liver damage. The combination of rapid drinking with the ability to study the motivational aspects in the same animal model of BD may be the most important hallmarks of an appropriate model of human BD behavior. Indeed, in humans, the study of the motivation for BD is an important component in the field of research on BD. Few animal models of voluntary alcohol intoxication in rats are available, and most of the models have been developed with inbred mice leading to difficulties with the evaluation of an interindividual vulnerability profile of BD. Important components of human BD behavior are the peer pressure and the social transmission. Social transmission of preference for ethanol has already been demonstrated in adolescent rats (Hunt et al., 2001).

These aspects need to be investigated in future studies in order to continue to improve the face validity of the BD models. Producing voluntary BD in animals is now possible and opens new perspectives for research. Future research is also needed regarding several unexplored aspects of BD behavior such as the construct validity (brain circuits and cognitive functioning), predictive validity (efficacy of pharmacotherapies), the role of gender, the role of social interaction and individual determinants such as impulsivity and genetic variations.

Legends of the figures

Figure 1. Number of publications found on PubMed on BD. Number of publications for 'BD and mice", BD and rats" and "BD and students" per year.

Figure 2. BEC profiles in humans, rats and mice after alcohol administration using oral intake, gavage of intra-peritoneal injections. BEC profiles after ethanol administration as a single dose of 0.5 or 0.75 g/kg of a 20% ethanol solution in either by means of intra-gastric gavage or intra-peritoneal injections in mice or rats. In humans acute binge ethanol was given as 2 ml vodka 40% v/v ethanol/kg body weight in a total volume of 300 ml orange/strawberry juice. Adapted from [START_REF] Bala | Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals[END_REF]Walker & Ehlers and Rose et al. (Walker and Ehlers, 2009[START_REF] Rose | Greater ethanol-induced locomotor activation in DBA/2J versus C57BL/6J mice is not predicted by presynaptic striatal dopamine dynamics[END_REF][START_REF] Bala | Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals[END_REF].

Figure 3 .

 3 Figure 3. Limiting the duration of session promotes binge drinking in the operant

Figure 4 .

 4 Figure 4. Pattern of alcohol deliveries in binge drinkers (14min access), high drinkers

Figure 5 .

 5 Figure 5. Hepatic gross morphology after BD. Representative liver harvested from

Figure 6 .

 6 Figure 6. Validity of the animal models of BD. Animal models of BD should display

Acknowledgements

BR & MN received a grant from the "Fonds Actions-Addictions" (http://actionsaddictions.org), which is an independent French foundation supporting evidencedbased actions against addictive disorders. MM received an invited-professor grant from the University of Picardie Jules Verne.

Table 1.

Title: Criteria for an animal model of BD.

Criteria for an animal model of BD 1-Voluntary intake / oral ingestion (without food deprivation or sweetener adulteration) 2-Quantity (>0.08g/dl) / visible signs of behavioral intoxication 3-Fast intake 4-Duration (repeated and at least subchronic) 5-Frequency / intermittence (few days off) 6-Brain damage/cognitive deficits and somatic damage (such as on liver) 7-Large inter-individual variability and possibility to investigate transition to dependence


Y Dendale

Prezado

Ludovic De Marzi
email: ludovic.demarzi@curie.fr

Catherine Nauraye

Pierre Lansonneur

Frédéric Pouzoulet

Annalisa Patriarca

Tim Schneider

Consuelo Guardiola

Hamid Mammar

Rémi Dendale

Yolanda Prezado

Spatial fractionation of the dose in proton therapy: Proton minibeam radiation therapy

Keywords: Proton, Minibeam radiation therapy, Pre-clinical research

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2

Purpose: In radiation therapy, a renewed interest is emerging for the study of spatially fractionated irradiation. In this review, a few applications using spatial fractionation of the dose will be discussed with a focus on proton minibeam radiation therapy (pMBRT). Examples of calculated dose (1D profiles and 2D dose distributions) and biological evidence obtained so far will be presented for various spatially 20 fractionated techniques GRID, MRT and MBRT. Recent results demonstrating that pMBRT leads to an increase in normal tissues sparing will be discussed, which opens the door to a dose escalation in the tumour and a possibly efficient treatment of very radioresistant tumours.

Introduction

Radiation-induced toxicity continues being a major concern in radiotherapy. There are ever increasing pieces of evidence of the influence of not only the dose, but the dose rate and the spatial fractionation of the dose in RT. Along this line, we are witnessing a renewed interest in spatially fractionated radiation therapy (SFRT), a strategy which has shown a remarkable capacity for tissue sparing. However, some elements indicate that the radiobiological response of tissues to very heterogeneous dose distributions might be poorly known. In this short review, a few applications using spatial fractionation of the dose will be discussed with a focus on proton minibeam radiation therapy (pMBRT).

The concept of spatially fractionated radiation therapy was firstly introduced at the beginning of the 20 th century with the aim of achieving a better skin sparing. Indeed the risk of skin necrosis was frequently reported when using poorly penetrating medium energy X-rays (≈60-70kV) to treat deep seated tumours.

The use of multi-perforated iron screens, proposed by A. Köhler (a German radiologist) in 1909 ( [START_REF] Köhler | Theorie einer Methode, bisher unmöglich unanwendbar hohe Dosen Röntgenstrahlen in der Tiefe des Gewebes zur therapeutischen Wirksamkeit zu bringen ohne schwere Schädigung des Patienten, zugleich eine Methode des Schutzes gegen Röntgenverbrennung überhaupt[END_REF]) and widely employed until the 1950s, helped reducing these adverse effects. The implementation was based on the collimation of an initial broad beam into a pattern of centimetre scale pencil beams to produce a nonuniform dose distribution. The healing of normal tissues was thus improved thanks to the presence of low dose irradiated tissues close to high dose areas in the pencil beam paths. The interest for SFRT declined with the advent of deeply penetrating megavoltage linear accelerators and their associated improved skin sparing due to dose build up effects. The use of SFRT was re-introduced in the early 1970s using 60 Co units ( [START_REF] Barkova | Theoretical calculation of the spatial distribution of a Co 60 gamma radiation dose field under a grid[END_REF][START_REF] Muth | Grid method in telecobalt therapy Radiobiol[END_REF]), and then in the 1990s under the name of GRID therapy ( [START_REF] Mohiuddin | Spatially fractionated (GRID) radiation for palliative treatment of advanced cancer[END_REF]). GRID therapy has been employed successfully in the palliative treatments of large and bulky tumours with early phase clinical trials having shown remarkable success. Recent reviews present clinical data and discuss new technological advances in GRID therapy ( [START_REF] Billena | A Current Review of Spatial Fractionation: Back to the Future?[END_REF][START_REF] Sheikh | Comparison of treatment planning approaches for spatially fractionated irradiation of deep tumors[END_REF]). One of the latest examples to date has been carried out with proton beams ( [START_REF] Gao | Spatially fractionated (GRID) radiation therapy using proton pencil beam scanning (PBS): Feasibility study and clinical implementation[END_REF]). In the early 1960s, studies on biological effects of cosmic radiation on mouses brains using deuterium beams of various sizes ( [START_REF] Zeman | Tolerance of mouse-brain tissue to high-energy deuterons[END_REF]) were conducted by the National Aeronautics and Space Administration (NASA) and provided first insights on the impact of small beams and dose-volume effects (the maximum tolerable doses increases as the irradiated tissue volumes is made smaller): a much higher tolerance to incredibly high radiation doses was observed when micrometer scale beams were used. For example, an incident dose of 4000 Gy to the mouse brain with a 25 µm beam showed no evidence of late tissue effect, whereas an incident dose of 140 Gy delivered by a 1 mm beam was sufficient to induce radionecrosis or damage to vessels.

Then, in the 1990s, Slatkin and colleagues ( [START_REF] Slatkin | Microbeam radiation therapy[END_REF]) profited from the advent of third generation synchrotron sources providing X-ray beams (X-rays are emitted tangentially from relativistic electron bunches circulating in a storage ring) with negligible beam divergence (<0.2 mrad) and high brilliance to propose Microbeam Radiation Therapy (MRT) ( [START_REF] Slatkin | Subacute neuropathological effects of microplanar beams of x-rays from a synchrotron wiggler[END_REF]). Synchrotron beams allow to obtain a pattern of peaks and valleys that remain fairly constant with tissue depth ( [START_REF] Bräuer-Krisch | Medical physics aspects of the synchrotron radiation therapies: Microbeam radiation therapy (MRT) and synchrotron stereotactic radiotherapy (SSRT)[END_REF]). MRT combines the spatial fractionation of the dose with extremely narrow beams (25-100 um). Indeed, the use of very narrow beams permits to maximally exploit the dose-volume effects [START_REF] Sheikh | Comparison of treatment planning approaches for spatially fractionated irradiation of deep tumors[END_REF]. Numerous experiments have then shown a spectacular increase in dose of tolerances of normal (skin, brain) tissues ( [START_REF] Bouchet | Preferential effect of synchrotron microbeam radiation therapy on intracerebral 9L gliosarcoma vascular networks[END_REF][START_REF] Dilmanian | Response of avian embryonic brain to spatially segmented X-ray microbeams[END_REF]). Because of the need of extremely high dose rates to maintain the patterns of peaks and valleys, this technique is mainly confined to large synchrotron light facilities. MRT is therefore today an experimental concept at the pre-clinical stage, and the translation into clinical phases is still under development.

A good compromise can be achieved with minibeam radiation therapy (MBRT) which uses slightly wider sub-millimetric field sizes (400-700 μm) than MRT, kilovoltage x-rays beams and spatial fractionation of the dose but offers technically easier realisation of dosimetry and positioning requirements ( [START_REF] Dilmanian | Interlaced x-ray microplanar beams: A radiosurgery approach with clinical potential[END_REF][START_REF] Prezado | A new method of creating minibeam patterns for synchrotron radiation therapy: a feasibility study[END_REF]).

Synchrotron-generated minibeams have first been shown to exhibit normal tissue sparing effects analogous to those obtained with MRT, and efforts were made to develop the use of non-synchrotron conventional small animal irradiators ( [START_REF] Bazyar | Lee Minibeam radiotherapy with small animal irradiators; in vitro and in vivo feasibility studies[END_REF][START_REF] Guardiola | Effect of X-ray minibeam radiation therapy on clonogenic survival of glioma cells[END_REF][START_REF] Prezado | Transfer of Minibeam Radiation Therapy into a cost-effective equipment for radiobiological studies: a proof of concept[END_REF]). Further improvements include the combination of charged particles, in particular with proton minibeam radiation therapy ( [START_REF] Prezado | Proton-minibeam radiation therapy: a proof of concept[END_REF]). Indeed, proton beams offer several advantages over X-rays MBRT, such as the precise ballistics of protons in the Bragg-peak region and a reduction of the integral dose. The multiple Coulomb scattering of charged particles also enables treatment of the tumour with a homogeneous dose distribution, while the normal tissues at shallow depth still benefit from spatial fractionation of the dose.

Proton minibeam radiation therapy

Proton therapy is a highly conformal technique that can maximize the dose gradient between tumour and surrounding structures. In contrast with photons, protons have the advantage of stopping at a given depth and reduced range straggling, resulting in a significant reduction of the integral dose to surrounding tissues. This technique has then been widely used for radioresistant tumours close to a sensitive structure or paediatric cancers, in which dose escalation in the target volume without increasing the dose to adjacent normal tissues is recommended ( [START_REF] Feuvret | Efficacy and Safety of Adjuvant Proton Therapy Combined With Surgery for Chondrosarcoma of the Skull Base: A Retrospective, Population-Based Study[END_REF]). The combination of the advantages of protons with MBRT is called proton minibeam radiation therapy (pMBRT). pMBRT has been recently implemented at a clinical facility (Orsay proton therapy center), in a clinically relevant environment using high energy beams (100 MeV). The biological experiments reported so far showed a remarkable increase in normal tissue sparing, both for the skin ( [START_REF] Girst | Proton Minibeam Radiation Therapy Reduces Side Effects in an In Vivo Mouse Ear Model[END_REF][START_REF] Prezado | Proton minibeam radiation therapy spares normal rat brain: Long-Term Clinical, Radiological and Histopathological Analysis[END_REF]) and brain ( [START_REF] Prezado | Proton minibeam radiation therapy spares normal rat brain: Long-Term Clinical, Radiological and Histopathological Analysis[END_REF]), along with an even higher control tumour effectiveness than standard therapy ( [START_REF] Prezado | Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy[END_REF]). pMBRT is still at the preclinical stage, but transition to the clinic is underway.

In pMBRT, two delivery techniques have been implemented for preclinical research ( [START_REF] Peucelle | Proton minibeam radiation therapy: Experimental dosimetry evaluation Medical physics[END_REF][START_REF] De Marzi | Implementation of planar proton minibeam radiation therapy using a pencil beam scanning system: A proof of concept study[END_REF]). One is based on passive scattering (PS) and the other delivery technique is based on Pencil Beam Scanning (PBS), combined with the use of multislit collimators. The latter consists of magnetically scanning several pencil beams over the target volume with the possibility of modulating the path, energy and intensity of the beams, and provides high dose-rates (up to several Gy/s), a potential reduction in neutron production compared to the passive scattering technique and significant improvements in the conformity of the dose distribution thanks to intensity modulation (IMPT). An example of a spread-out Bragg peak and a dose distribution that can be obtained with pMBRT and scanned pencil beams is shown Figure 1: by means of intensity modulation of Bragg peaks, spatial fractionation is maintained at shallow depths while homogeneous dose distribution is obtained around the target.

Dosimetric properties

Contrary to conventional RT which provides homogeneous dose profiles, the lateral dose profiles in MBRT consist of peaks and valleys (see Figure 2). The peak-to-valley-dose ratio (PVDR) is a dosimetric parameter defined as the ratio of the peak dose over the valley dose which is frequently used to evaluate the potential sparing of normal tissues. High PVDR values and low valley doses are required to ensure tumour control and healthy tissue sparing respectively ( [START_REF] Dilmanian | Response of rat intracranial 9L gliosarcoma to microbeam radiation therapy[END_REF]). The distance separating two minibeams centers is known as the centre-to-centre distance (c-t-c). GRID therapy typically uses large pencil beams (1 cm diameter), whereas MRT and MBRT use sub-millimetric beams of 25-100 µm and 400-700 µm respectively. GRID therapy uses arrays of beams separated by centre-to-centre (c-t-c) distances of a few cm, whereas MRT and MBRT typically use c-t-c distances from 200 to 400 µm and 2 to 6 mm respectively. Most spatially fractionated set-ups use 1D planar beams arrangements, but 2D lattice structures have also been proposed. Examples of calculated lateral dose profile for various spatially fractionated techniques GRID, MRT and MBRT) are shown in Figure 2. Two clinical examples of dose distributions that can be obtained with GRID and pMBRT plans using a proton PBS technique are also shown in Figure 3 for a treatment of a glioma patient.

Preclinical evidence

Normal tissue dose tolerances and tumour growth delay have been observed in several recent biological experiments. The first long term evaluation of the effects of pMBRT on normal brain was performed by Prezado et al ([27]). In this work, the whole brain of Fischer rats was irradiated at a dose close to the maximum brain tolerance dose (25 Gy) within a single broad beam conventional irradiation, or received pMBRT irradiation with peak doses of 58 Gy (corresponding to a mean dose of 25 Gy). The animals were followed up for 6 months. A magnetic resonance imaging (MRI) study using a 7-T small-animal MRI scanner was performed along with a histological analysis. Rats treated with conventional proton irradiation exhibited severe moist desquamation, permanent epilation and substantial brain damage. In contrast, rats in the pMBRT group exhibited no skin damage, reversible epilation and no brain damage. A good sparing of the skin was also observed in an independent experiment by a German team ( [START_REF] Girst | Proton Minibeam Radiation Therapy Reduces Side Effects in an In Vivo Mouse Ear Model[END_REF]), who reported on reduced side effects in the mouse ear after pMBRT with 20 MeV protons.

In a second research study, the evaluation of the tumour effectiveness of pMBRT for the treatment of RG2 glioma-bearing was performed ( [START_REF] Prezado | Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy[END_REF][START_REF] Prezado | Proton minibeam radiation therapy widens the therapeutic window for high-grade gliomas[END_REF]) showing superior or equivalent tumour control effectiveness compared to standard proton therapy. RG2 glioma bearing rats were irradiated, receiving conventional proton therapy or pMBRT with either a homogeneous or a heterogenous dose distribution in the tumour (25 Gy in one fraction were delivered). The whole brain (excluding the olfactory bulb) was irradiated with peak doses of 70 Gy and a mean dose of 30 Gy. A significant proportion of long-term survivals (> 6 months) free of tumour was obtained, reaching in some cases 67 %. No significant lesions were observed.

The results of the experiments performed so far with very heterogeneous spatial distributions, such as with GRID/MRT/MBRT, indicate the activation of different biological mechanisms (which might not be specific to proton beams) from those involved when direct damage to DNA as a primary target by ionizing radiation takes place. However, the biological basis of tissue response to spatial fractionation is not yet completely understood, and some of the biological results obtained so far clearly suggest the participation of distinct biological mechanisms: in particular, dose-volume effects (migration of undamaged cells to repair the radiation damage [START_REF] Hopewell | Volume effects in radiobiology as applied to radiotherapy[END_REF]), cell signalling effects such as cohort effects (signalling between irradiated cells within an irradiated volume [START_REF] Asur | High dose bystander effects in spatially fractionated radiation therapy[END_REF]) or bystander cellular responses (signalling between irradiated cells and nearby undamaged/unirradiated cells [START_REF] Dilmanian | Tissue-sparing effect of x-ray microplanar beams particularly in the CNS: Is a bystander effect involved?[END_REF][START_REF] Bj | Sykes Radiation-Induced Bystander Effects: What Are They, and How Relevant Are They to Human Radiation Exposures?[END_REF]), the activation of the immune system ( [START_REF] Bouchet | Early Gene Expression Analysis in 9L Orthotopic Tumor-Bearing Rats Identifies Immune Modulation in Molecular Response to Synchrotron Microbeam Radiation Therapy[END_REF]), or the role of the vasculature (prompt vascular repair effect, [START_REF] Bouchet | Effects of microbeam radiation therapy on normal and tumoral blood vessels[END_REF]) among others were all identified as being involved in the healing process of radiation damage.

Discussion and conclusion

Some recent results demonstrate that GRID/MRT/pMBRT leads to an increase in normal tissues sparing, which opens the door to a dose escalation in the tumour, and possibly an efficient treatment of very radioresistant tumours. In particular, the use of protons instead of photons for pMBRT seems to be advantageous because of a more localised release of the dose. The normal tissue dose tolerances and delay of tumour growth observed in recent biological experiments also showed a remarkable gain and good rates of control. In addition, spatial fractionation may allow hypofractionated treatment of tumours as it spares healthy tissues at shallow depths ( [START_REF] Dilmanian | Minibeam therapy with protons and light ions: Physical feasibility and potential to reduce radiation side effects and to facilitate hypofractionation[END_REF]). On the other hand, very short irradiation times and hypofractionated schemes are prerequisite to MBRT since any movement or misalignment of the healthy tissue would result in smearing and deterioration of the required dose distribution. In these conditions, the linear-quadratic model may not be sufficiently accurate to compare SFRT and conventional radiotherapy for large doses per fraction, and the definition of tolerance doses as well as an optimisation of dose-time treatment protocols remain an important topic that needs to be fostered to advance pre-clinical work towards clinical trials.

Figure 1 .

 1 Figure 1. Example of a calculated spread-out Bragg peak and a dose distribution that can be obtained with 190 pMBRT and pencil beam scanning.

Figure 2 .

 2 Figure 2. Example of a calculated lateral dose profile in spatially fractionated techniques for classical GRID, MRT and MBRT techniques.

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Figure 3 :

 3 Figure 3: Example of planned dose distribution of GRID and pMBRT using a proton PBS technique for the treatment of a glioma patient.

Figure 2 200Figure 3

 23 Figure 2 200

Acknowledgements

Our work was supported by Institut National du Cancer (INCa), DGOS, Cancéropôle Ile de France (grant agreement 2015-1-RT-06).

Conflicts of interest

The authors have no relevant conflicts of interest to disclose.