Experimental and Clinical Psychopharmacology
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Translational dynamics of alcohol tolerance of preclinical models and human laboratory studies
Carolina L. Haass-Koffler
1,2,3*
, Nazzareno Cannella
3
and Roberto Ciccocioppo
3
1
Center for Alcohol and Addiction Studies, Department of Psychiatry and Human Behavior, Warren Alpert
Medical School and Department of Behavioral and Social Sciences, School of Public Health, Brown University,
Providence, RI;
2
Section on Clinical Psychoneuroendocrinology and Neuropsychopharmacology, National
Institute on Alcohol Abuse and Alcoholism Division of Intramural Clinical and Biological Research and National
Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Bethesda, MD;
3
School of
Pharmacy, Pharmacology Unit, University of Camerino, Italia.
Abstract
Increasing sensitivity due to alcohol intake has been explored using molecular and cellular mechanisms of
sensitization, adaptive biobehavioral changes, and through negative experiences of altered function during
withdrawal. However, within both a preclinical and human laboratory setting, little has been elucidated towards
understanding the neural substrates of decreased sensitivity to alcohol effects, i.e. alcohol tolerance. More
paradigms assessing alcohol tolerance are needed. Tolerance can be assessed through both self-reported
response (subjective) and observed measurements (objective). Therefore, sensitivity to alcohol is an
exploitable variable that can be utilized to disentangle the diverse alcohol use disorder (AUD) phenotypical
profile. This literature review focuses on preclinical models and human laboratory studies to evaluate alcohol
tolerance and its modulating factors. Increased understanding of alcohol tolerance has the potential to reduce
gaps between preclinical models and human laboratory studies to better evaluate the development of alcohol-
related biobehavioral responses. Furthermore, alcohol tolerance can be used as an AUD phenotypic variable
in randomized clinical trials (RCTs) designed for developing AUD therapies.
Keyword
Alcohol use disorder; tolerance; human laboratory studies; preclinical models; translational research
Public Significance Statements
This review article focuses on the translational efforts utilized to understand the neural substrates that affect
decreased sensitivity to the effects of alcohol, i.e. alcohol tolerance. The field of alcohol research requires
enhanced translational efforts to develop paradigms that can be utilized to elucidate the diverse alcohol use
disorder phenotypical profile.
2
Acknowledgements
Dr. Haass-Koffler is supported by the National Institute on Alcohol Abuse and Alcoholism (K01AA023867;
R01AA027760: R01 AA026589) and in part by the National Institute of General Medical Sciences (NIGMS),
Center of Biomedical Research Excellence (COBRE, P20 GM130414). Dr. Ciccocioppo is supported by the
program PRIN (Progetti di Rilevante Interesse Nazionale) 2017 of the Italian Ministry of Education University
and Research (project 2017SXEXT5). Dr. Cannella is supported by The European Foundation for Alcohol
Research (ERAB) research program 2018 (project EA1840).
All authors have contributed to the manuscript in a significant manner, and all authors have read and
approved the final manuscript.
The authors have no conflict of interest related to this work. There are no relationships between these
authors and financial supporters or other organizations that could have influenced this research.
We thank Zoe E Brown for her contribution to the editing of the manuscript.
This is a review article. No data on human subjects or animals were used for this manuscript.
3
1. Introduction
The desire to consume and the capacity to metabolize alcohol extends deep into human history. Roughly
9000 years ago, humans learned to ferment foods (Schrago, Menezes, Moreira, Pissinatti, & Seuanez, 2012;
Steiper & Young, 2006), however, the most ancient form of alcohol dehydrogenase class IV (ADH4), the
enzyme in the digestive tract that is capable of metabolizing alcohol, was found in hominoid lineage as far back
as 70 million years ago (Carrigan et al., 2015).
Increased sensitivity to alcohol has been explored using multiple approaches, including molecular and
cellular mechanisms of sensitization (Hoek & Pastorino, 2004), adaptive alcohol-related bio-behavioral
changes, and through negative experiences of altered function during withdrawal (Gilpin & Koob, 2008).
However, there is limited research aimed at understanding the neural substrates of the decreased sensitivity to
alcohol’s effects (i.e. alcohol tolerance) within a preclinical and human laboratory setting.
Measuring alcohol tolerance is a complex task, due to the fact that it can be differentiated in a variety of
ways; for review see: (Kalant, 1998). For example, metabolic tolerance results from the induction of alcohol
within metabolizing enzymes resulting in changes in the pharmacokinetic profiles (Ryan et al., 1985). Acute
tolerance refers to tolerance developed during the alcohol administration procedure (within session), chronic
tolerance is reached through multiple or prolonged alcohol exposures (between sessions), and rapid tolerance
is described as the process that occurs after a second alcohol exposure followed by complete clearance of the
first dose during which, the second dose is given between 8-24 hours after the initial dose (Khanna, Chau, &
Shah, 1996).
Observations conducted on dogs one hundred years ago, revealed a more pronounced alcohol-induced
ataxia on the alcohol ascending limb compared to the descending limb (Mellanby, 1919). Decades later, Wistar
rats, after a single administration of alcohol (1.0-2.8 g/kg dose), portrayed significant motor function impairment
for a given blood level on the falling (descending) compared to the rising (ascending) limb of the blood alcohol
curve (LeBlanc, Kalant, & Gibbins, 1975). Interestingly, one of the first models for alcohol use disorder (AUD)
was based upon the fact that, after alcohol withdrawal, rats who were injected with alcohol were much less
responsive to subsequent injection of alcohol compared to control rats who were previously exposed only to
water (Cicero, Snider, Perez, & Swanson, 1971). This early model demonstrated that it is possible to test the
classic criteria signifying AUD by evaluating physical dependence and tolerance to alcohol. In humans,
tolerance can be assessed by both self-reported responses and objective measurements. As such, it
represents an exploitable variable in the field of alcohol research. Alcohol tolerance can be utilized towards
understanding the complex AUD phenotype and developing novel pharmacotherapies (Haass-Koffler &
Perciballi, 2020).
2. Functional, metabolic and cross tolerance
Tolerance to alcohol can result from pharmacodynamic and/or pharmacokinetic effects. Pharmacodynamic (or
functional) tolerance refers to neurobiological and/or neurochemical adaptations that reduce functional
4
impairment (i.e. cognitive and motor). In addition to CNS adaptation, chronic alcohol consumption leads to
metabolic adaptation, which is characterized by an increase in alcohol metabolism and rate of blood alcohol
clearance in individuals without liver disease (Cederbaum, 2012). In brief, the disposition of alcohol (as for any
other xenobiotic) is characterized by four criteria: absorption, distribution, metabolism and excretion (ADME);
for extensive review see: (Jones, 2019). Metabolic tolerance refers to changes in alcohol pharmacokinetic
effects due to changes in metabolism and elimination (B. Tabakoff, Cornell, & Hoffman, 1986).
First pass metabolism of alcohol occurs in the stomach and liver (Lim et al., 1993). A small part portion
of ingested alcohol undergoes oxidation in the stomach and will not enter into the systemic circulation
(Cederbaum, 2012). The liver is primarily responsible for the rate of enzymatic oxidation during first pass
metabolism (Swift, 2003), however, it is import to know that first pass metabolism of alcohol is determined by
the speed of gastric emptying (Oneta et al., 1998).
Some of the suggested mechanisms leading to metabolic tolerance to alcohol includes induction of
alcohol dehydrogenase (ADH), induction of cytochrome P450 2E1 (CYP2E1), increased re-oxidation of
nicotinamide adenine dinucleotide (NADH) by mitochondria, increased cytokines, and hypoxia of hepatocytes
(origin of alcohol toxicity) (Cederbaum, 2012). Also, alteration of alcohol elimination rates may contribute to the
development of metabolic tolerance. The major enzyme for metabolizing alcohol, hepatic alcohol
dehydrogenase, produces acetaldehyde (and NADH) which is further oxidized to acetate. Chronic alcohol
consumption decreases acetaldehyde oxidation, either due to decreased aldehyde dehydrogenase 2 Family
Member (ALDH2) activity or impaired mitochondrial function. As a result, metabolic adaptation, characterized
by elevated circulating levels of acetaldehyde, observed in individuals with AUD may result in an increased
production and or decreased removal of acetaldehyde. Additionally, oxidation of alcohol by CYP2E1 are
induced by alcohol and represent additional pathways to eliminate alcohol especially at high concentrations.
Other potential mechanisms underlying metabolic tolerance are extensively reviewed in (Cederbaum, 2012).
The contribution of the elimination rates impact on the development of metabolic tolerance is based on
several biochemical processes and is supported by preclinical studies. For example, studies evaluating alcohol
elimination rates comparing Wistar and alcohol preferring P rats on chronic free-choice drinking and forced
alcohol feeding, showed that all the alcohol preferring P rats exposed to alcohol by either free-choice or forced-
feeding exhibited increased alcohol elimination rates (Lumeng & Li, 1986).
Metabolic tolerance has also been evaluated in flies. In Drosophila, alcohol metabolizing enzyme
alcohol dehydrogenase, ADH, is characterized by variation between the two alleles Adh
F
(more active)
and Adh
S
, (less active). Larvae with a deficiency of the ADH gene are very sensitive to alcohol toxicity (David,
Bocquet, Arens, & Fouillet, 1976), however, there was not a phenotypic difference in alcohol-induced sedation
within locomotor activity when compared to wild-type controls (Singh & Heberlein, 2000).
In addition to functional and metabolic tolerance, alcohol tolerance can be assed via cross-tolerance,
which depends on similarities in the pharmacological profile of alcohol and other drugs, such as sedatives (e.g.
barbiturates) and anxiolytics (e.g. benzodiazepines). Cross-tolerance appears to follow the trajectory of
5
functional rather than metabolic tolerance; for extensive review see: (B. Tabakoff et al., 1986). First, the
changes in ADH activity would not be expected to produce metabolic cross-tolerance to other drugs (Koivula &
Lindros, 1975). Additionally, studies in rats consuming alcohol and barbiturates found that the development of
tolerance and impairments in motor control tasks were characterized by unaltered phenobarbital elimination
from the serum, suggesting that cross-tolerance was due to CNS adaptations (Lau, Tang, & Falk, 1981).
Studies with alcohol and benzodiazepines (chlordiazepoxide) reported a partial and short-lasting development
of tolerance, suggesting different neuronal pathways between the drugs (Chan, Schanley, Aleo, & Leong,
1985).
3. Alcohol tolerance in Drosophila melanogaster
Previous research using flies have described tolerance through alcohol’s impact on the development of larvae
and adult fly survival (Guarnieri & Heberlein, 2003; Kaun, Devineni, & Heberlein, 2012). Adult Drosophila
melanogaster are a particularly useful model to elucidate mechanisms of alcohol tolerance since they do not
have changes in enzymatic ADH levels after developing rapid tolerance (Geer et al., 1988). The short life cycle
of a fly may also help to evaluate the development from rapid tolerance to chronic tolerance without the
variability introduced by metabolic changes.
Studies with Drosophila melanogaster have contributed to our understanding of alcohol-related
behaviors in other preclinical models and humans, suggesting that alcohol tolerance may be conserved across
evolution (Petruccelli & Kaun, 2019). One of the most astounding discoveries of alcohol tolerance using
Drosophila melanogaster models involves the role of stress in alcohol-related behaviors. The development of
alcohol tolerance in flies relies on two distinct molecular pathways. The first pathway involves the octopamine
system (an organic chemical in invertebrates that is related to vertebrates’ noradrenaline), which is specific to
the development of chronic alcohol tolerance. Flies with a mutation in the Tbh gene (the encoding tyramine -
hydroxylase enzyme that converts tyramine to octopamine) developed reduced tolerance compared to wildtype
flies even after a single sedating alcohol pre-exposure; for review see: (Monastirioti, 1999).
The other pathway is related to the newly discovered hangover gene which encodes a large nuclear
zinc-finger protein required for cellular stress response (Scholz, Franz, & Heberlein, 2005). After heat shock
(stress-induction), flies with the hangover mutation developed rapid tolerance to alcohol, i.e. instead of
exhibiting a sedating effect during alcohol pre-exposure, they expressed tolerant behavior as if they had
previously been exposed to alcohol. Additional data examining stress-tolerant outcomes was tested using flies
bred specifically for alcohol resistance. Similar to the flies with the hangover mutation, flies bred for alcohol
tolerance exhibited increased resistance to stressors (heat shock, desiccation and chemicals) (Cohan &
Hoffmann, 1986). As the flies became tolerant to stressors, they began to show greater resistance during
alcohol exposure (Hoffmann & Parsons, 1989).
Additional neurotransmitters tested in flies have also portrayed similar effects in vertebrates. For
example, the -aminobutyric acid (GABA) B antagonist CGP54626 has shown to decrease sedation in flies
6
(Dzitoyeva, Dimitrijevic, & Manev, 2003). Similar responses have been observed in alcohol-mediated behavior
within rat models (Maccioni & Colombo, 2009).
Among the preclinical models used to test alcohol tolerance, the Drosophila melanogaster offers
numerous advantages recently explored in greater context. The fly mushroom body is comprised of a neuropil
structure required for processing memories and learning; for review see: (Petruccelli & Kaun, 2019) that have
been involved in not only alcohol-induced hyperactivity (King et al., 2011), but also in alcohol tolerance (Engel
et al., 2016). This work suggests that flies regulate alcohol tolerance, a form of behavioral plasticity, via
conserved signal transduction pathways by anchoring signaling molecules to the plasma membrane in
proximity to the actin cytoskeleton (Parkhurst et al., 2018).
4. Alcohol tolerance in rodent models
Alcohol tolerance has been evaluated in rodent models (Erwin & Deitrich, 1996; Ponomarev & Crabbe, 2002).
Behaviorally, high drinking rats have consistently demonstrated a greater degree of tolerance to alcohol's
intoxicating effects; for review see: (Kalant, 1998). One of the original studies on acute alcohol tolerance in
rodent models was conducted by simultaneous measurements of arterial blood and brain alcohol level in male
Wistar rats. This study demonstrated that the brain alcohol level is in equilibrium with the arterial alcohol level
(LeBlanc et al., 1975). These results were also confirmed later in human studies that evaluated the effects of
alcohol on pharmacokinetic profiles within breath alcohol concentration (BrAC), and venous and arterial blood
concentrations after oral consumption (Martin, Moll, Schmid, & Dettli, 1984). This study demonstrated that,
during the absorption process, BrAC follows the trajectory of arterial blood. Due to this trajectory, BrAC levels
are a more accurate prediction of arterial blood concentrations compared to venous blood alcohol levels.
However, during elimination, BrAC, and arterial and venous blood follow similar trajectories.
A more systematic approach to evaluate alcohol tolerance was initiated with the development of the
Alko Alcohol (AA) and Alko Nonalcohol (ANA) rat lines by the ALKO, the State Alcohol Monopoly of Finland (K.
Eriksson, 1971). After the development of the AA and ANA rat lines, alcohol tolerant (AT) and alcohol
nontolerant (ANT) rats were selectively bred to measure acute and chronic tolerance (Kalervo Eriksson & Rusi,
1981). Both lines have been utilized to elucidate mechanisms that affect alcohol-induced motor impairment.
The AT rats show decreased sensitivity to alcohol induced motor impairment on a tilting plane over a wide
range of alcohol doses, without difference in other behavioral measures for alcohol sensitivity. Consistently, the
AA rats portrayed increased rapid and chronic tolerance to alcohol-induced hypothermia, and exhibited a more
profound effect in motor impairment and sleep patterns than the ANA rats (Le & Kiianmaa, 1988).
A second generation of rodents with the same phenotype were bred and tested for ataxia, loss of
righting reflex (LORR), and blood ethanol concentration at regain of the righting reflex (BECRRR) at the
University of Colorado Alcohol Research Center (Radcliffe et al., 2004). Ataxia was measured on the inclined
plane at 5 and 30 minutes after an intraperitoneal alcohol dose of 2 g/kg. The AT rats developed acute
tolerance compared to NAT rats. In addition, LORR and BECRRR, following an alcohol dose of 3.5 g/kg, were
7
tested as a proxy of acute alcohol tolerance. The AT rats had a significantly higher BECRRR compared to the
NAT rats. There was however, no difference in LORR (Radcliffe et al., 2004).
Later, a study tested the hypothesis that the larger voluntary alcohol intake of the AA strain might be
due not only to a stronger innate (genetically linked) tolerance to alcohol, but may also be linked to a greater
likelihood of developing tolerance after chronic alcohol exposure (Nikander & Pekkanen, 1977). After chronic
alcohol administration, both strains increased their tolerance, but the AA rats exhibited higher rates of
tolerance compared to the ANA rats (Nikander & Pekkanen, 1977). Overall, the alcohol-preferring rats (P rats)
develop tolerance to alcohol more quickly, and are less sensitive to the sedative-hypnotic effects of alcohol
compared with non-preferring animals; for extensive review see: (McBride & Li, 1998). Motor impairment (tilt-
plane) and hypothermia tests were adopted to characterize the differences between rapid alcohol tolerance
and chronic alcohol tolerance. A series of experiments with control rats (those who were not exposed to
alcohol or the apparatus) demonstrated similarities between the mechanisms of rapid and chronic tolerance
(Khanna et al., 1996).
The development of alcohol tolerance as a way of detecting persistent rapid changes in the effects felt
due to alcohol were demonstrated in male Swiss mice (Crabbe, Rigter, Uijlen, & Strijbos, 1979). Research
utilizing hypothermia as a measure of alcohol physical dependence (Ritzmann & Tabakoff, 1976),
demonstrated that mice develop tolerance to the hypothermic effects of a single alcohol injection upon
administration of an equivalent dose 24 hours prior to the initial administration. Blood alcohol concentration did
not differ in tolerant and nontolerant mice, and tolerance was present within 10 minutes of the second alcohol
injection; this phenomenon was later termed rapid tolerance, a measure that is unique to the previously coined
term metabolic tolerance (Crabbe et al., 1979).
The effect of stress on acute alcohol tolerance was also tested in mice using yohimbine, an 2 receptor
blocker. Yohimbine was able to antagonize acute tolerance in mice during the rolling test drum, suggesting that
2 receptors may play an important role in mediating acute alcohol tolerance (Edwards, Schabinsky, Jackson,
Starmer, & Jenkins, 1983). In addition, the intraventricular administration of 6-hydroxydopamine, a neurotoxic
for noradrenergic and dopaminergic neurons, in mice prior to chronic exposure to alcohol prevented the
development of acute tolerance (B Tabakoff & Ritzmann, 1977). These findings suggest that the role of the
noradrenergic system plays a role in the development of acute tolerance, and can provide key neurobiological
mechanisms signifying underlying acute tolerance. This insight, along with knowledge of the role of the
noradrenaline analogue octopamine in rapid alcohol tolerance among flies, as reviewed above, confirms the
role of this noradrenergic system in tolerance-related mechanisms.
In rodent models, the role of the neurotransmitter, neuromodulator and neuroendocrine systems in the
context of alcohol tolerance have been predominately focused on acute and rapid tolerance. For example, the
original AT rat lines exhibited a higher capacity to develop acute tolerance with dysregulation of the
noradrenergic and GABAergic systems (Kiianmaa & Hellevuo, 1990). Within the GABA-ergic system, studies in
male Swiss mice showed that the GABAB agonist baclofen is capable of blocking rapid tolerance, while
8
GABAB antagonists (CCGP36742 and CGP56433) facilitate the development of rapid tolerance in a dose
dependent manner (Zaleski, Nunes Filho, Lemos, & Morato, 2001). Actions due to alcohol exposure at the
GABAA receptor are also influenced by endogenous neuroactive steroids. A pharmacological study showed
that pretreatment with pregnenolone (a neurosteroid) among female mice significantly facilitated the
development of rapid tolerance (Barbosa & Morato, 2001) and the stimulatory action of pregnenolone was
reversed by the inhibitory action of muscimol (a GABAA agonist). Rodent studies focusing on the GABA-ergic
system reveled potential links between neurosteroids and the development of tolerance. However,
neurosteroid studies have cast mixed results, as they can block or facilitate chronic tolerance (within a time
span of 2-5 days). In the rota-rod apparatus, the impairing effect of alcohol occurred on the fifth day of
treatment of epipregnanolone (an endogenous steroid that acts as a negative allosteric modulator of the
GABAA receptor and reverses the effect of allopregnanolone). This effect was enhanced by pretreatment with
pregnenolone (endogenous steroid and precursor of most steroid hormones), on the second day (Barbosa &
Morato, 2000).
The role of the opioid system in the development of rapid tolerance has been evaluated using
pharmacological probes in male Wistar rats (Varaschin, Wazlawik, & Morato, 2005) by infusion of selective -
(naloxonazine), - (naltrindole), and - (nor-binaltorphimine) opioid antagonists in the core and shell of the
nucleus accumbens. The results of this study suggest that -opioid receptors in both the shell and core of the
nucleus accumbens participate in the modulation of rapid tolerance to alcohol. However, it is also possible that
the -opioid played a role in this development, but this role has been limited to the accumbens’ core (Varaschin
& Morato, 2009).
The role of the N-methyl-D-aspartate (NMDA) system on rapid alcohol tolerance was tested by
intraperitoneal injection of the active isomer (+) of the NMDA antagonists MK-801(dizocilpine) and ketamine in
Swiss female mice. Administration of (+)MK-801 and ketamine blocked the development of rapid tolerance as
measured in the tilt-plane apparatus (Barreto, Lemos, & Morato, 1998).
There is a large body of preclinical research that supports the role of oxytocin in alcohol tolerance; for
extensive review see: (Pedersen, 2017). Oxytocin administered (intraperitoneally in mice and subcutaneous in
rats) 10 min to 2 hours before each alcohol daily dose over 3 to 5 days significantly inhibited tolerance
formation as measured by hypothermia and sedation (Jodogne, Tirelli, Klingbiel, & Legros, 1991; Puciklowski,
Kostowski, & Trzaskowska, 1985; Szabó, Kovács, Székeli, & Telegdy, 1985).
Finally, conflicting results on motor impairment and anticonvulsant effects due to alcohol intake have
suggested differences among both sexes and between male and female hormones when assessing alcohol
tolerance in rats (Koirala, Alele, & Devaud, 2008).
5. Alcohol tolerance in Rhesus macaques
Research examining alcohol tolerance in nonhuman primates allows scientists to dose and evaluate behaviors
indicative of alcohol response in a preclinical model very similar to human tendencies. Primates’ large brains
9
have allowed researchers to utilize magnetic resonance spectroscopy (MRS) to detect decreases in brain
membrane alcohol partitioning as a way to measure the development of alcohol tolerance (Kaufman et al.,
1994).
Innate sensitivity and the use of different doses prompts investigation of both rapid tolerance (Kalant,
1993; Khanna et al., 1996) and sensitization to the acute effects of alcohol (Schwandt, Higley, Suomi, Heilig, &
Barr, 2008). As observed in mice with hypothermia (Crabbe et al., 1979), Rhesus macaques ataxic behavior
decreased from dose one to dose two, while locomotor stimulation increased between doses. This change in
behavior was independent of blood alcohol concentrations and was not associated with the amount of time
between doses (roughly 5-30 days occurred between alcohol administration) (Schwandt et al., 2008). Results
from this work suggest that Rhesus macaques are capable of developing rapid tolerance to motor impairing
and locomotor sensitization. A factor analysis showed that the behavioral response (exhibited through the
development of rapid tolerance) was evident within a single dose of alcohol, and rapid tolerance may persist
for some time following the first alcohol dose (Schwandt et al., 2008). Studies in rodents have cast similar
results. Locomotor sensitization in mice lasts 30 to 60 days (Fish, DeBold, & Miczek, 2002; Lessov & Phillips,
1998) and rapid tolerance in alcohol preferring P rats persists for 10 days (Gatto et al., 1987).
Rhesus macaques, have a long period of adolescence (similar to human populations) as such, they
offer opportunities to evaluate alcohol-related phenotypes during youth; for review see: (Schwandt et al.,
2010). Furthermore, Rhesus macaques studies can extend evaluation of the genetic variations associated with
alcohol tolerance in a controlled laboratory setting without introduction of additional environmental variables. In
humans, decreased sensitivity to alcohol has been demonstrated as a predictor of AUD, and variations in the
gene-linked polymorphic region of the serotonin transporter (5-HTTLPR) have been associated with neuronal
mechanisms responsible for alcohol tolerance (Hinckers et al., 2006; Turker et al., 1998). Rhesus macaques
homozygous for the rh5-HTTLPR, showed a decreased sensitivity to the ataxic and sedating effects of alcohol
after being intoxicated by intravenous administration of alcohol (with doses of 2.2 g/kg for males and 2.0 g/kg
for females): this 21-basepair length variation has been detected within the transcriptional control region in
human beings (Barr et al., 2003). However, the phenotypic expression of this genotype is environmentally
dependent since peer-reared adolescent animals predicted lower intoxication scores compared to mother-
reared animals (Barr et al., 2003).
6. Alcohol tolerance in human laboratory studies
Human studies measuring subjective responses to alcohol have greatly contributed to our understanding of the
development of acute alcohol tolerance (Trim, Schuckit, & Smith, 2009). However, the concept of subjective
response is not novel since it was previously evaluated as a clinical predictor of AUD risk (Schuckit & Smith,
1996). The development of AUD paradigms to evaluate acute alcohol tolerance have risen from multiple
models with inclusion of both subjective and objective measures, for review see (Haass-Koffler & Perciballi,
2020). Those paradigms have been based on the low level response model (LLR), which focuses on the
10
hypothesis that individuals who are less responsive to the sedative effects of alcohol are at greater risk of
developing AUD (Schuckit, 1994), and the differentiator model, which evaluates behavioral responses during
alcohol’s biphasic effects (Newlin & Thomson, 1990).
Intravenous alcohol administration paradigms have been utilized to evaluate alcohol tolerance. These
paradigms allow for a highly controlled alcohol concentration procedure, and can limit the effects felt due to
variability in pharmacokinetics (Ramchandani, Bolane, Li, & O'Connor, 1999). Intravenous alcohol paradigms
(which bypass absorption and first pass metabolism) focus on pharmacokinetic principles, and allow
researchers to examine short and long term tolerance within human laboratory studies through a quantitative
approach by controlling for BrAC exposure over the entire procedure (Ramchandani et al., 2006). Recent
intravenous alcohol administration studies evaluating the role of hangovers post alcohol consumption have
demonstrated that individuals who are heavier drinkers display signs of chronic tolerance more frequently than
those who consume less, suggesting that a direct relationship between hangovers, tolerance development,
and alcohol consumption may exist (Vatsalya, Stangl, Schmidt, & Ramchandani, 2018).
Another important paradigm to consider when translating preclinical models to human laboratory
studies is the environment (Ciccocioppo, 2012). Additionally, within clinical research, it has been observed that
a novel drinking environment may elucidate a more stimulating experience compared to a familiar place
(Plebani et al., 2012). The development of a bar-like laboratory has provided an additional setting to determine
predictors of alcohol-related behavior where many variables can be controlled in a more naturalistic
environment compared to the typical “sterile” clinical laboratory setting (Fox et al., 2012; Haass-Koffler et al.,
2017; Haass-Koffler, Leggio, Davidson, & Swift, 2015; Kenna et al., 2016; Thomas, Bacon, Sinha, Uhart, &
Adinoff, 2012). Additionally, mechanisms that impact knowledge acquisition play a critical role in impaired
behavior (Vogel-Sprott, 1979). Accordingly, pre-drug cues determine conditioned preparatory responses to
counteract the substance’s effects leading to tolerance (Poulos & Cappell, 1991; Siegel, 1989). As such, it is
important to acknowledge that individuals tend to lose inhibition when drinking in unfamiliar places when
assessing tolerance within a laboratory setting. In fact, alcohol tolerance can facilitate a drinker’s ability to
anticipate the effects of alcohol. This aspect of human alcohol tolerance suggests that tolerance is not a static
state, but it is subject to environmental stimuli (Ostling & Fillmore, 2010).
Ecological Momentary Assessment (EMA) sampling (Shiffman, Stone, & Hufford, 2008) has the
advantage of assessing alcohol-related behaviors in real time within a natural environment. However, while the
EMA real time assessments may have numerous advantages, compared to retrospective calendar methods
utilized to assess drinking outcomes (Morgenstern, Kuerbis, & Muench, 2014), the EMA has some limitations.
EMA data is collected in the absence of the experimenter therefore, may lack objectivity when assessing
alcohol tolerance.
The rate at which an individual’s blood alcohol concentration rises should be taken into consideration in
order to understand the intra-individual differences in acute tolerance in the laboratory setting. Greater
psychomotor impairment occurs on the ascending limb while a reduced impairment is observed on the
11
descending limb. For example, with a faster rise in blood alcohol concentration, there is a consequent increase
in psychomotor impairment (Mark T Fillmore, Vogel‐Sprott, & Research, 1998). Thus, the rate at which blood
alcohol concentration rises may predict impaired behavior, rather than the blood alcohol concentration value
previously detected. Intravenous alcohol administration techniques have great relevance to the concept of
assessing acute alcohol tolerance and the rate at which alcohol concentration changes. The control of many
variables provide consistent exposure rates during the alcohol biphasic profile (Ramchandani et al., 1999) to
assess Mellanby differences in motor control, cognition and memory between the ascending and descending
limbs (Mellanby, 1919).
Sex should be considered when measuring alcohol tolerance within the laboratory setting. It is well
known that pharmacokinetically, women metabolize (Kwo et al., 1998; Li, Beard, Orr, Kwo, & Ramchandani,
1998) alcohol differently than men. Women reached significantly higher peak blood alcohol concentrations than
men when alcohol was administered based on individual body weight, however, there was no difference
detected between the sexes when alcohol was administered based on total body water (Goist & Sutker,
1985). Unfortunately, limited research on the effects of sex hormones on alcohol tolerance and behavioral
response has been conducted (Mumenthaler, Taylor, O'Hara, & Yesavage, 1999).
Additional intra-variabilities affecting measurements of alcohol tolerance have been explored among
human subjects. For example, nicotine may impact the biphasic effects of alcohol, such as, enhancing its
positive effects (e.g., stimulation) (Kouri, McCarthy, Faust, & Lukas, 2004) and diminishing its sedative effects
(Perkins et al., 1994). This is an important aspect considering that smoking is highly prevalent among
individuals with AUD (Kalman, Morissette, & George, 2005).
Finally, little is known about the mechanisms that re-establish control following impaired behavior. Both
the diminishing and stimulating aspects of behavioral control appear to be affected by alcohol (M. T. Fillmore,
Marczinski, & Bowman, 2005). Interestingly, acute alcohol tolerance seems to develop within impaired
activation but not with impaired inhibition (Fillmore, Marczinski et al. 2005). Evidence of acute excitatory
responses, but not of inhibitory responses suggests some degree of independence between these two
mechanisms of control (Logan, 1994; Logan & Cowan, 1984). Further studies are needed to better understand
the mechanisms underlying recovery of behavioral control.
7. Conclusion
It has long been established that the positive reinforcing effects of alcohol during initial alcohol consumption,
followed by chronic alcohol exposure, result in neuroadaptations that can eventually lead to transient and
prolonged neuroplasticity that contributes to the development of AUD; for review see: (Koob & Le Moal, 2008).
This change in brain biochemistry has been observed via the signaling of neurotransmitters, receptor proteins,
and neuronal connectivity; for review see: (Kalivas & O'Brien, 2008). To date, most of the literature on
12
preclinical models used for measuring alcohol tolerance is not only scarce, but outdated, however, different
preclinical models employed to test alcohol tolerance have offered diverse advantages.
The Drosophila melanogaster models have demonstrated that there are two independent cellular
pathways that affect rapid and chronic tolerance; one pathway involves the octopamine (leading to rapid
tolerance) and the other pathway is independent of octopamine signaling (leading to chronic tolerance) (Scholz
et al., 2005), suggesting that stress plays different roles in the development of alcohol tolerance. One of the
most important studies on alcohol tolerance in rodent models resulted from simultaneous measurements in rat
arterial blood and brain levels (LeBlanc et al., 1975). This study elucidates the equilibrium process of the
arterio-venous differences in alcohol concentration.
The long adolescent period of the Rhesus Macaques has offered an opportunity to evaluate alcohol-
effects comparable to human adolescence, a time often deemed the age at which humans may begin
developing alcohol tolerance (Schwandt et al., 2010). Due to the lack of data available, early literature on
rodent models has revealed limitations in assessing sex differences among alcohol tolerance. GABAA
receptors are influenced by endogenous neuroactive steroids affecting different alcohol rates between the
sexes (Barbosa & Morato, 2001). As such, sensitization to GABAA by allosteric compounds (e.g.
benzodiazepines) have been shown to influence (through the role of cross-tolerance) alcohol use disorder;
GABAA sensitization may represent an exploitable variable utilized to study gender and sex hormone
differences in the molecular effects of alcohol on the development of tolerance (Grobin, Matthews, Devaud, &
Morrow, 1998). Furthermore, to our knowledge, limited work regarding this topic has been conducted in
humans (Mumenthaler et al., 1999).
Human bar laboratory studies offer the advantage of testing alcohol tolerance in a more naturalistic
setting rather than the typical “sterile” clinical laboratory setting; however, non-bar laboratory challenge
paradigms examining tolerance and sensitization to alcohol have and will continue to be significant contributors
to the study of tolerance.
In conclusion, both preclinical models and human studies offer opportunities to study the
pharmacokinetic effects of tolerance (alterations of alcohol metabolism) and pharmacodynamic effects of
alcohol tolerance (CNS adaptation) that often lead to alcohol related diseases (Cederbaum, 2012). Alcohol
tolerance is a complex measure, but may represent one additional variable that can be utilized to expand
understanding of the diverse AUD phenotypical profile and prompt development of new medications to treat
alcohol use disorder.
13
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