The Effect of High-Fructose Corn Syrup on Ethanol Taking, Seeking, and Palatability in Rats

by

Samantha Ayoub

A Thesis Presented to

The University of Guelph

In partial fulfilment of requirements for the degree of

Master of Science in Psychology + Neuroscience

Guelph, Ontario, Canada

© Samantha Ayoub, August, 2018

ii

ABSTRACT

THE EFFECT OF HIGH-FRUCTOSE CORN SYRUP ON ETHANOL TAKING, SEEKING AND PALATABILITY IN RATS

Samantha Ayoub Advisor: University of Guelph, 2018 Dr. Francesco Leri

Many alcoholic beverages contain sweeteners, such as high-fructose corn syrup

(HFCS), which has been implicated in increasing alcohol consumption and encouraging

hazardous drinking patterns. Therefore, four experiments were designed to investigate

whether the addition of HFCS to an ethanol (EtOH) solution could alter its intake,

hedonic taste, and sensitivity to food deprivation stress in Sprague Dawley rats.

Experiments 1 and 2 revealed that HFCS caused a robust increase in EtOH intake

during operant intraoral self-administration (IO SA), however an equally palatable, yet

non-caloric sweetener (saccharin) could not replicate this effect. This suggests the

caloric value of a sweetener mediates the effect of sweeteners on EtOH intake.

Experiments 3 and 4 revealed food deprivation stress increased EtOH consumption

during resumption following extinction, but only when HFCS was added to the solution.

The palatability of 10% EtOH and 25% HFCS was also monitored across IO SA, and

under conditions of food deprivation stress. It was found that the palatability of 10%

EtOH and 25% HFCS increased following acquisition. More interestingly, food

deprivation stress only increased HFCS palatability. Thus, it could be the heightened

palatability of HFCS under stressful conditions that contributes to increased operant

responding for solutions containing HFCS during resumption in rats. These results have

implications for the use of highly palatable and caloric sweeteners in EtOH beverages

because of the impact they may have on excessive EtOH intake.

iii

ACKOWLEDGEMENTS

I would like to thank my advisor, Dr. Francesco Leri, for the opportunity to

complete my thesis in an astounding laboratory. Your mentorship and guidance

throughout this process helped me develop into a better scientific researcher and

academic writer. Your intellect and passion for this field of work is admirable to say the

least.

I would like to thank my thesis committee member, and practicum advisor, Dr.

Linda Parker. It has been my pleasure to work in your lab, even if so briefly.

Additionally, your support and mentorship throughout my thesis was well recognized,

and I cannot thank you enough. Lastly, I would like to thank the members of the Parker

laboratory who helped train me and made me feel welcome; especially Erin Rock.

I would like to thank all my labmates for their continued support and

encouragement throughout the past two years. I could not have asked for a better group

of individuals to work with. In particular, I would like to thank Meenu Minhas for training

me and getting me started up in the Leri laboratory. I will truly miss contacting you when

I am stuck in a rut and am not sure what to do next. I appreciate all your help so much.

Finally, I would like to thank my family and friends. You have all been so positive

and supportive throughout my entire academic career. I love you all so much.

iv

Table of Contents ABSTRACT…………………………………………………………………………………… ii Acknowledgements…………………………………………………………………………… iii List of Figures…………………………………………………………………………………. v General Introduction………………………………………………………………………….. 1 Manuscript………………………………………………………………………………......... 20 Abstract……………………………………………………………............................ 21 Introduction……………………………………………………………………………. 22 Methods………………………………………………………………………………. 25 Statistical analyses…………………………………………………………….......... 32 Results………………………………………………………………………………… 32 Discussion…………………………………………………………………………….. 36 References……………………………………………………………………………. 43 Figure legends……………………………………………………………………….. 53 Figures……………………………………………………………………………….. 56 General Discussion………………………………………………………………………….. 60 Summary of results………………………………………………………………….. 60 Methodological considerations…………………………………………………….. 60 Future directions…………………………………………………………………….. 62 Conclusion…………………………………………………………………………… 64 References…………………………………………………………………………………… 66

v

LIST OF FIGURES Figure 1……………………………………………………………………………………….. 56 Figure 2……………………………………………………………………………………….. 57 Figure 3……………………………………………………………………………………….. 58 Figure 4……………………………………………………………………………………….. 59

1

General rationale and summary of experiments

Heavy episodic drinking is prevalent in youth and has been shown to elevate the

risk of developing an alcohol use disorder later in life (Chassin, Pitts, & Prost, 2002).

Furthermore, youth who display such drinking patterns prior to the age of 18 are less

likely to seek help, and have increased risks for chronic relapsing following treatment

(Hingson, Heeren, & Winter, 2006). It has been suggested that the use of sweeteners in

alcoholic beverages reduces the aversive taste of alcohol in naïve drinkers, and leads to

increased consumption (Mosher & Johnsson, 2005; Lanier, Hayes, & Duffy, 2005;

Romanus, 2000; Copeland, Stevenson, Gates, & Dillon, 2007). In fact, youth that

primarily consume sweetened alcoholic beverages have four times the risk of

experiencing alcohol-related injuries and report a higher frequency of alcohol binges

(Albers, Siegel, Ramirez, Ross, DeJong, & Jernigan, 2015). Although this may seem

intuitively obvious, the mechanisms by which sweeteners increase alcohol intake are

not fully understood. More specifically, sweeteners not only change the taste of alcohol,

but they also support reliable effects on behaviour and the brain. Thus, sweeteners also

have the potential to synergistically alter alcohol’s reinforcing value because they elicit

approach and conditioned behaviour (Yasoshima & Shimura, 2015; Sclafani & Ackroff,

2017; White & Carr, 1985; Agmo & Marroquin, 1997), and maintain high levels of

operant self-administration (Levy et al., 2015; Avena, Long, & Hoebel, 2005). Recently,

our laboratory has explored the behavioural and neurobiological responses to high-

fructose corn syrup (HFCS) in rats (Levy et al., 2014, Levy et al., 2015; Daniels,

Marshall & Leri, 2016; Levy et al., 2018). In comparison to sucrose (which contains 50%

glucose and 50% fructose), HFCS has a higher proportion of fructose (typically, 55%

2

fructose), and has been linked to several adverse health effects including obesity, heart

disease, and poor metabolic health (Meyers, Mourra, & Beeler, 2017; Bray, 2013;

Stanhope et al., 2015). The findings from our lab suggest that HFCS has strong

reinforcing properties, and could possibly be a more potent behavioural reinforcer than

sucrose. HFCS is highly prevalent in our diets, and can be found in many of the

sweetened alcoholic beverages available for purchase (e.g. Palm Bay, Bacardi Silver,

Smirnoff Ice, Mike’s Hard Lemonade). Thus, the current experiments were designed to

investigate the effect of HFCS on ethanol (EtOH) intake, palatability, and persistence of

operant responding in Sprague Dawley rats.

Experiment 1 used an operant intraoral self-administration (IO SA) technique to

explore whether HFCS could alter EtOH intake. It was found that HFCS produced

robust increases in IO SA behaviour and EtOH intake.

In Experiment 2, a similar experimental design was used to assess the effect of an

equally palatable, yet non-caloric sweetener (saccharin; SACC) on EtOH intake. It was

found that SACC increased EtOH intake, however this effect was minimal in comparison

to what was observed by the addition of HFCS. These results suggest that the caloric

value of a sweetener powerfully mediates the effect of sweeteners on EtOH intake in

rats.

Experiment 3 tested the persistence of operant responding following behavioural

extinction in rats trained to self-administer 10% EtOH in 25% HFCS. This experiment

also included a stress manipulation, because stress is known to alter the reinforcing

effects of drugs of abuse and facilitate drug-seeking in many species (Shaham, Erb, &

Stewart, 2000). The stress selected was food deprivation because it has been shown to

3

have potent effects on animal drug-seeking behaviours (Shalev, Yap, & Shaham, 2001;

Carroll, 1985) and because of the relevance to the behaviour being explored (i.e.,

ingestion of calories from both EtOH and HFCS). It was found that food deprivation

stress increased persistent operant responding for EtOH, only when it was mixed in

HFCS.

In Experiment 4, EtOH and HFCS taste reactivity was measured throughout the IO

SA of EtOH + HFCS to assess how the palatability of these solutions are altered over

subsequent exposure periods, and under conditions of food deprivation stress. This

revealed two key findings. First, rats produced more palatable responses to EtOH and

HFCS following IO SA acquisition. Second, food deprivation stress increased palatable

responding to HFCS, but did not alter responding to EtOH. Thus, it could be the

increased palatability of HFCS under stressful conditions that contributes to the

increased operant responding for solutions containing HFCS during resumption

following extinction in rats. In other words, the motivation to consume an EtOH

beverage under normal or stressful conditions can be increased by the addition of

HFCS.

Collectively, these results have implications for the use of highly palatable and

caloric sweeteners in EtOH beverages because of the impact they have on EtOH intake

in naïve drinkers. Below, I will sequentially review the key aspects of my research.

Heavy Episodic Drinking in Youth Increases the Risk of AUD Development

Heavy episodic drinking refers to the consumption of 60 or more grams of ethanol

within a single occasion, occurring at least once per month (World Health Organization,

2014). This quantity of alcohol roughly equates to 5 standard drinks for men, and 4

4

standard drinks for women. Worldwide, heavy episodic drinking patterns are the most

prevelant in young and often underaged drinkers (World Health Organization, 2014). A

nationwide Canadian survey revealed 14.7% of youth aged 15-17 reported these

consumption patterns, while this number increased to 33.4% between respondents

aged 18-19 (Archie, Zangeneh-Kazemi, & Akhtar-Danesh, 2012). Furthermore, the US

National Institute on Alcohol Abuse and Alcoholism (2015) estimates 1.45 million youth

aged 12 to 17 ‘binge’ on alcoholic beverages at least once per month. These statistics

are particularly concerning because young drinkers characterized as ‘bingers’ have an

exceedingly elevated risk for subsequent alcohol use disorders (AUDs) compared to

‘non-binger’ youths (Chassin, Pitts, & Prost, 2002). Furthermore, early onset of alcohol

drinking is correlated with increased risks of subsequent alcohol misuse and associated

problem behaviours in adulthood (Gruber, DiClemente, Anderson, & Lodico, 1996;

Higson, Heeren, & Winter, 2006; DeWit, Adlaf, Offord, & Ogborne, 2000; Hawkins,

Graham, Maguin, Abbott, Hill, & Catalano, 1997). Thus, reducing excessive alcohol

consumption in young, naïve drinkers could be an effective strategy to mitigate the high

prevalence of disordered drinking worldwide.

Alcohol Use Disorders

Alcohol use disorders increase many health risks for the abuser and place a heavy

burden on healthcare systems worldwide. In fact, the World Health Organization (2009)

reported alcohol use as the third leading risk factor contributing to the global burden of

disease. Heavy alcohol consumption is causally linked to liver and heart disease, birth

defects, cancer, and unintentional injuries (Rehm et al., 2009). Additionally, AUDs are

comorbid with a wide range of psychiatric disorders, including, anxiety disorders, major

5

depressive disorders, post-traumatic stress disorder, substance use disorders, and

schizophrenia (Petrakis, Gonzalez, Rosenheck, & Krystal, 2002; Regier et al., 1990).

Despite the negative consequences associated with excessive alcohol consumption, the

prevalence rate for AUDs remains high at approximately 4% worldwide (The World

Health Organization, 2014).

(1) Chronic Relapse

The high occurrence of AUDs is partially attributable to the inability of abusers to

maintain abstinence following alcohol detoxification and in-patient treatment programs.

Studies reveal high relapse rates following both short-term (Moos & Moos, 2006) and

long-term remission periods (Jin, Rourke, Patterson, Taylor, & Grant, 1998), signifying

that a relapse to excessive alcohol consumption can be triggered regardless of long-

term periods of abstinence. For example, Jin et al., (1998) measured relapse rates in

long-term abstainers, defined as individuals with 18-months of stable abstinence, and

found 31% had relapsed prior to an 11-year follow-up period.

The high and frequent occurrence of relapses during recovery has led to the

recognition of AUDs as chronic relapsing disorders. Given that the recovery from an

AUD is a long and sometimes impossible road, preventative measures may be an

effective method to reduce AUD prevalence. That is, preventing disordered drinking in

naïve drinkers may reduce the incidents of AUDs.

(2) Triggers of Relapse

Identifying AUDs as chronic relapsing disorders has drawn attention to the

identification of factors that provoke a relapse, and the neural correlates that underlie

this behaviour. Exposure to alcohol-paired stimuli is one determinant of alcohol relapse

6

following abstinence (Perry, Zbukvic, Kim, & Lawrence, 2014). For example, detoxified

alcoholics experience increased alcohol craving, and physiological responses, when

exposed to alcohol-paired stimuli in a laboratory setting (Garland, Franken, & Howard,

2012; Cooney, et al., 1997). Furthermore, the magnitude of post-treatment reactivity to

alcohol-paired stimuli accurately predicts future relapses (Garland et al., 2012). These

findings suggest alcohol-paired stimuli possess incentive salience, acquired through

repetitive pairings with alcohol’s reinforcing pharmacological properties. That is, through

associative learning, alcohol-paired stimuli become conditioned cues, or conditioned

reinforcers (CRs), capable of eliciting a craving response and motivating behaviour.

Given the taste of alcohol is prominent during consumption, alcohol-paired taste cues

may act as CRs to motivate alcohol consumption behaviour. Indeed, exposure to the

taste of beer significantly increases alcohol craving in heavy drinkers when compared to

other flavoured controls (i.e. Gatorade; Oberlin et al., 2013). Additionally, neuroimaging

studies reveal that the taste of alcohol activates mesocorticolimbic structures in the

brain (Filbey et al., 2008), and increases dopamine (DA) release in these areas (Oberlin

et al., 2013). Given that mesolimbic DA signaling is implicated in reward, motivation

(see Robins & Everitt, 1996 for a review) and goal-directed behaviour (Wise & Rompre,

1989; Horvitz, 2000), these findings suggest alcohol-paired tastes can drive appetitive

behavioural responding for alcohol, and perhaps contribute to relapse following

abstinence.

Exposure to stress is another major determinant of relapses following AUD treatment

(Preston & Epstein, 2011). For example, the occurrence of severe stress following

treatment is correlated with increased relapse risk (Brown, Vik, Patterson, Grant, &

7

Schuckit, 1995; Brown et al., 1990). Brown et al. (1990) found alcoholics who had

relapsed during a three-month treatment follow-up had self-reported significantly more

severe stress during the study period than those who remained abstinent. Additionally,

stress induces alcohol cravings in abstinent alcoholics (Preston & Epstein, 2011). Fox,

Bergquist, Hong, and Sinha, (2007) used a stress imagery technique to induce anxiety

in former alcoholics within a laboratory setting. They found stress imagery significantly

increased alcohol craving, as well as measures of blood pressure, in comparison to a

neutral imagery condition. Similar to cue-induced craving, the magnitude of laboratory

stress-induced alcohol craving can also predict abstinence following treatment (Sinha et

al., 2011; Higley et al., 2011). For example, Higley et al. (2011) demonstrated greater

laboratory stress-induced craving was associated with a significantly shorter duration of

alcohol abstinence, fewer total days of abstinence, and a higher mean number of drinks

per week following treatment.

In sum, both exposure to alcohol-paired stimuli acting as CRs, and stress, have been

implicated in provoking alcohol relapse following AUD treatment. The data presented is

largely derived from human laboratory studies of alcohol-induced craving, or from

correlative measures of CR/stress exposure on subsequent relapse rates. Animal

models of relapse can causally measure the influence of CRs and stress on relapse-like

responding, thereby strengthening the assumptions made regarding CR and stress

exposure on relapse vulnerability. Operant drug self-administration (SA) procedures

model “drug-taking” and “drug-seeking” in animals, and are used to analyze the factors

that contribute to drug addiction, including relapse-like responding (Panlilio & Goldberg,

2007).

8

Measuring Drug-Intake in Animals: Operant Self-Administration Models

The fundamental assumptions used by the operant SA model date back to the early

works of Thorndike (1898) who proposed behaviours that produce favourable outcomes

are more likely to be repeated than behaviours producing non-favourable outcomes.

Using this theoretical framework, operant SA procedures allow an animal to perform a

behavioural response for the administration of drug reinforcement; thus, if the drug is

indeed a reinforcing substance, the operant response which led to its administration

should continue to be repeated.

In rodents, operant drug SA models are conducted in operant conditioning chambers

that are typically equipped with an active and inactive lever (O’Conner, Chapman,

Butler, & Mead, 2011). Responding on the active lever (a lever-press) results in the

delivery of drug reinforcement, thereby producing a measure of drug-reinforced

responding, or “drug-taking”. A lever-press on the inactive lever results in no

programmed consequences, and can therefore be used as a control measure of non-

reinforced operant responding. Measures of drug intake (number of drug infusions) or

operant responding (active versus inactive lever responding) are used as indications of

SA behavior, and help researchers make inferences about the drug’s abuse potential.

(1) Extinction-Reinstatement Procedures

Extinction-reinstatement procedures stem from operant drug SA models, and

measure “drug-seeking” in animals (Panlilio & Goldberg, 2007; Shaham, Shalev, Lu,

deWitt, & Stewart, 2002). Following initial acquisition of SA, extinction of the drug-taking

behaviour is accomplished by removing response-contingent drug reinforcement. As a

result, responding under extinction conditions declines rapidly as the behaviour is no

9

longer being reinforced (Katner, Magalong, & Weiss, 1999). Thus, any behavioural

responding in the absence of drug reinforcement is a measure of “drug-seeking” rather

than drug-taking. Once drug-seeking is extinguished, a reinstatement test can be used

to delineate the factors that provoke a reinstatement of the extinguished responding (i.e.

drug-seeking) for that drug. Therefore, extinction-reinstatement procedures can

measure relapse-like responding following a period of abstinence and are invaluable

tools in drug addiction research (Panlilio & Goldberg, 2007; Martin & Weiss, 2013).

In corroboration with the human literature on relapse vulnerability, exposure to

alcohol-paired stimuli reinstates extinguished alcohol-seeking in rats. For example, re-

exposure to drug-taking environments potently reinstates previously extinguished drug-

taking behaviour (see Crombag, Bossert, Kova, & Shaham, 2008 for a review).

Chaudhri, Sahuque, Cone and Janak (2008) trained rats to self-administer alcohol in an

environmental context distinguished by distinctive visual, olfactory and tactile stimuli.

Subsequently, alcohol-taking behaviour was extinguished in a separate and

perceptually altered context. During reinstatement, animals were given a small oral

alcohol prime (i.e. re-exposure to oral alcohol) in the drug-taking context, or in the

extinction context. Reinstated responding for alcohol was observed when rats were

primed in the alcohol-taking context but not in the extinction context; thus,

demonstrating alcohol-paired contextual stimuli modulate behavioural responding for

alcohol after periods of abstinence.

The taste of alcohol, or flavours paired with alcohol, also hold conditioned reinforcing

effects that affect alcohol-seeking behavior in rats. For example, oral alcohol primes

(Bienkowski, Koros, Kostowski, & Bogucka-Bonikowska, 2000; Maccioni et al., 2007)

10

and alcohol-paired taste cues (Knight et al., 2016) reinstate responding for alcohol

following behavioural extinction. Knight et al. (2016) paired a distinct flavour with alcohol

during SA acquisition to create an excitatory conditioned stimulus (CS+). Therefore, the

CS+ signaled the presence of alcohol reinforcement while rats acquired and stabilized

alcohol-taking behaviour. Subsequently, a different flavour was paired with water during

an extinction phase to create an inhibitory conditioned stimulus (CS-), which served to

signal the absence of alcohol reinforcement. During a reinstatement session, rats were

placed back into the conditioning chambers and allowed to self-administer either the

CS+, CS- or a neutral taste stimulus (CS0) in water. Lever responding was significantly

elevated in the CS+ group when compared to extinction baseline responding,

demonstrating a flavour previously paired with alcohol reinstated extinguished alcohol-

seeking behaviour in rats. Moreover, there was no reinstatement of lever responding

found in the CS- or CS0 group. Taken together, these studies support the human

literature that suggests the conditioned reinforcing properties of alcohol-paired cues,

such as taste, provoke appetitive behavioural responding for alcohol following periods of

abstinence.

Exposure to stress also reinstates alcohol-seeking behaviour in rats using the

extinction-reinstatement model. Lê, Harding, Juzutsch, Funk and Shaham (2005)

induced a stressed-state in rats by exposing animals to 10-minutes of intermittent

footshock (0.8 mA). Footshock stress immediately preceding a reinstatement test

potently increased alcohol-seeking behaviours following extinction. Moreover, stress

may interact with the effect of conditioned cue exposure to potentiate alcohol-seeking

behaviours. For example, Liu and Weiss (2002) demonstrated a brief exposure to

11

footshock stress exacerbated alcohol-seeking behaviours elicited by conditioned cue

exposure in comparison to alcohol-seeking behaviours elicited by conditioned cue

exposure alone.

(2) Extinction-Reacquisition Procedures

Extinction-reacquisition procedures also stem from SA models to measure the

persistence of drug-taking following the extinction of this behaviour. During reacquisition

testing, response reinforcement is restored, making it possible to explore the capacity

for an animal to relearn extinguished operant responding. Typically, the reacquisition of

extinguished operant drug-reinforced responding, otherwise called resumption, is more

rapid in comparison to the original acquisition (Bouton, Winterbauer, & Todd, 2012;

Willcocks & McNally, 2013). This suggests that the extinction of a reinforced behaviour

does not equate with the unlearning of previously reinforced responding. Moreover,

extinction-reacquisition procedures also support the notion that exposure to CRs and/or

stress exacerbates relapse-like responding. For example, Yu, Chen and Sharp (2014)

demonstrated exposure to restraint stress during a period of abstinence exacerbated

the reacquisition of nicotine self-administration when animals were reintroduced to the

operant chambers.

In sum, the same factors that have been implicated to provoke relapse in human

populations (i.e. alcohol-paired cues, stress) also reinstate extinguished alcohol-seeking

and alcohol-taking behaviours in rats using animal models of relapse. This signifies the

translational efficacy of using operant alcohol SA procedures in animals to make

inferences about human alcohol relapse behaviour. Furthermore, by producing alcohol-

taking and alcohol-seeking behaviour in animal models, the neurobiology of this

12

responding can be investigated by pharmacological manipulation of different

neurotransmitter systems. Understanding the neural mechanisms underlying alcohol-

taking and alcohol-seeking behaviour will assist in the development of therapeutic

treatment options for AUD patients who experience chronic relapses following

treatment.

Food Deprivation Stress

Food deprivation increases behavioural and neurobiological biomarkers of stress in

humans and animals. For example, the magnitude of dietary restriction is associated

with increased urinary cortisol excretion (McLean, Barr, & Prior, 2001) and elevated

salivary cortisol levels (Anderson, Shapiro, Lundgren, Spataro, & Frye, 2002) in healthy

adults. Rodent studies corroborate these findings by showing food deprivation increases

corticosterone levels (Nowland, Hugunin, & Rogers, 2011; Dietze, Lees, Fink, Brosda &

Voigt, 2016). Thus food deprivation elicits a stress response that could produce reward-

seeking behaviour in humans and animals.

In humans, being in a food deprived state is associated with increases in motivation

to seek rewarding stimuli. For example, Leeman, O’Malley, White, and McKee (2010)

found that daily smokers who participated in a 12-hour food deprivation period smoked

more cigarettes during the study period and reported stronger cravings for nicotine than

participants who were satiated. Moreover, studies support that food deprivation

increases a healthy individual’s persistence of behavioural responding for rewarding

stimuli (Hogenkamp, Shechter, St-Onge, Sclafani, & Kissileff, 2017; Bulik & Brinded,

1994). For example, food deprivation has been found to increase the amount of

behavioural responding for sweetened beverages (Hogenkamp, Shechter, St-Onge,

13

Sclafani, & Kissileff, 2017; Bulik & Brinded, 1994) and cigarettes (Bulik & Brinded,

1994).

In animal models, food deprivation stress reliably increases drug-taking and

drug-seeking behaviours. For example, 24- and 48-hour food deprivation has been

shown to potently increase heroin-seeking and cocaine-seeking in rodents (Shalev,

Yap, & Shaham, 2001; D’Cunha, Sedki, Macri, Casola, & Shalev, 2013; Tobin,

Newman, Quinn, Shalev, 2009; Carroll, 1985; Highfield, Mead, Grimm, Rocha, &

Shaham, 2002). Furthermore, animal researchers frequently use food deprivation as a

reliable method to increase the intake of various drugs of abuse (Carr, 2007).

The Self-Administration of Alcohol in Rats

Rats will self-administer alcohol intravenously (IV; Smith & Davis, 1974; Gass &

Olive, 2007), intragastrically (IG; Waller, McBride, Gatto, Lumeng, & Li, 1984; Fidler &

Clews, 2006) or orally (Chaudhri et al., 2008). IV and IG self-administration are useful

tools in studies of drug addiction, however their use in alcohol-related research is limited

by the lack of face-validity provided when applying their findings to the prototypical oral

consumption patterns found in humans. Additionally, IG and IV methods by-pass

important orosensory aspects of alcohol consumption (such as smell and taste), which

as previously mentioned, have been demonstrated to modulate alcohol craving in

humans (Oberlin et al., 2013) and alcohol-seeking behaviours in rats (Katner,

Magalong, & Weiss, 1999; Maccionia et al., 2007; Knight et al., 2016).

Although oral alcohol SA in rats can be established, there are procedural

complications with the existing system which delivers alcohol reinforcement using a fluid

receptacle (Fl-R). More specifically, a dipper-cup raises a small quantity (0.1 ml) of an

14

alcohol solution from a fluid reservoir, into a trough within the operant conditioning

chamber (Chaudhri, et al., 2008; Maccioni et al., 2007). The dipper-cup presents the

alcohol solution for a predetermined time interval, and then recedes back into the Fl-R

until the next reinforcement is delivered. Therefore, this approach requires an animal to

perform the operant response, and then move to the Fl-R to consume the alcohol

reinforcer. As such, the Fl-R delivery approach poses several caveats for measuring

operant alcohol SA responding.

One limitation of the Fl-R delivery approach is the delay experienced between the

operant response and delivery of reinforcement; a variable known to retard SA

acquisition (Williams, 1976; Black, Belluzzi, & Stein, 1985). Williams (1976) measured

the influence of varying response-reinforcement delays on the responding for grain in

food-deprived pigeons. It was demonstrated that even at the shortest delay (3-secs)

pigeons displayed a drastic reduction in responding when compared to a non-delayed

baseline. Additionally, Black, Belluzzi and Stein (1985) found delays as minimal as 1-

sec significantly impaired the acquisition of reinforcing brain self-stimulation in rats.

Thus, the delay experienced between the performance of an operant response and the

delivery of an oral alcohol reinforcer into a Fl-R may produce deficits in alcohol SA

acquisition, and lower behavioural responding.

Another limitation of the Fl-R approach is the inability to accurately measure alcohol

intake on a trial-by-trial basis, limiting the interpretation of consumption data. That is,

following each alcohol presentation the dipper-cup recedes back into the fluid reservoir

without measuring whether the liquid was consumed. The amount of alcohol intake can

be calculated by measuring the remaining liquid in the fluid reservoir at the end of each

15

session; however, trial-by-trial consumption is left undetermined. Additionally, without

trial-by-trial consumption data it is unknown whether each operant response is being

reinforced, thereby devaluing the capacity for SA behaviour to be a reliable measure of

alcohol-reinforced responding. Furthermore, unreinforced responding can lead to an

extinction of the operant response (Katner, Magalong, & Weiss, 1999), hence disturbing

the acquisition and maintenance of SA behaviour.

An additional shortcoming of the Fl-R technique is the variability in timing of liquid

prime deliveries. Oral alcohol primes are often delivered during

reinstatement/reacquisition procedures, as they have been demonstrated to reinstate

extinguished alcohol-seeking in rats (Bienkowski et al., 2000; Maccioni et al., 2007).

Using the Fl-R technique, an effective oral prime delivery is contingent upon the

animal’s approach response to the Fl-R. However, a rat must be responsive to the Fl-R

to receive the administered prime. If rats differ in their receptiveness to the Fl-R, the

timing of obtaining the oral prime could vary between-subjects. It has been

demonstrated that as many as 15 dipper-cup presentations are required to reinstate

extinguished responding for alcohol in rats (Bienkowski et al., 2000), an effect perhaps

attributable to inconsistencies in prime delivery between-subjects.

A Novel Technique Approach to the Oral Self-Administration of Alcohol in Rats.

Recently, a novel intraoral (IO) delivery technique was developed to administer liquid

reinforcement directly into the oral cavity of a rat during operant SA sessions (Levy et

al., 2014). By surgically implanting an oral cannula into the cheek, the IO delivery

technique overcomes many of the caveats posed by the Fl-R approach. First, IO

administrations will strengthen the response-reinforcement contingency by rapidly

16

delivering alcohol reinforcement upon the behavioural response. Second, in IO SA rats

directly consume each liquid reinforcer, providing consumption data for each trial. Third,

IO SA permits passive IO liquid infusions, allowing for a priming technique that does not

require movement toward the Fl-R, thereby decreasing between-subject variability.

In addition to strengthening the measurement of operant alcohol SA responding, IO

SA allows alcohol’s taste properties to be more efficiently investigated during

reinstatement and reacquisition procedures. That is, rather than the presentation of a

liquid prime in a Fl-R (visual + orosensory cues), IO primes are delivered directly into

the oral cavity to permit the analyses of taste and orosensory stimulation selectively.

The Reinforcing Properties of Sweeteners

Sweet substances have substantial and reliable effects on behavior. For example,

people describe the taste of sweeteners as pleasant (Thai et al., 2011; Frank et al.,

2008) and show preference for beverages that have been sweetened (Tatzer, Schubert,

Timischl, & Simbruner, 1985; Ventura & Mennella, 2011). In addition, individuals display

the motivation to consume sweetened beverages by exerting effort to gain access to its

administration (Schebendach et al., 2017; Guttman, 1954). The liking and preference for

sweeteners may be linked to activity in cortical regions that process reward functions

(Frank et al., 2008). Indeed, a variety of sweeteners increase the release of dopamine

into the nucleus accumbens (NAc; Rada, Avena & Hoebel, 2005; Wheeler et al., 2011;

Hajnal, Smith, & Norgren, 2004), a brain region implicated in attributing incentive

salience to rewarding stimuli (Wyvell & Berridge, 2000; Steinberg et al., 2014).

Although individuals show difficulty in discriminating between caloric and non-caloric

sweetness (Frank et al., 2008), caloric sweeteners are known to be more potent

17

reinforcers in terms of their behavioural and neurobiological effects (Domingos et al.,

2011; White & Carr, 1985). Some have even argued that caloric sweeteners may have

“addictive-like” properties, similar to drugs of abuse (Levy et al., 2015; Lenoir, Serre,

Cantin, & Ahmed, 2007; Madsen & Ahmed, 2014).

(1) High fructose corn syrup

High-fructose corn syrup (HFCS) is highly prevalent in our diets (Barlow, McKee,

Basu, & Stuckler, 2017; Bray, Nielsen, & Popkin, 2004), and has been linked to several

adverse health effects including obesity, heart disease, and poor metabolic health

(Meyers, Mourra, & Beeler, 2017; Bray, 2013; Stanhope et al., 2015). In comparison to

sucrose (which contains 50% glucose and 50% fructose), HFCS has a higher proportion

of fructose (typically, 55% fructose), and this is sufficient to engender different

behaviours during operant intraoral self-administration, as well as different patterns of

gene expression in the brain (Levy et al., 2015). Despite the recognition that the

reinforcing properties of HFCS differs from that of sucrose, (Ackroff & Sclafani, 2011;

Levy et al., 2015) research investigating the effect of HFCS on behaviour and brain is

quite limited.

Recently, this laboratory has explored behavioral and neurobiological responses

to high-fructose corn syrup in laboratory rats (HFCS; Levy et al., 2014; Levy et al., 2015;

Levy et al., 2018; Daniels, Marshall, & Leri, 2016). In Levy et al. (2015), rats were

trained to intraorally self-administer isocaloric solutions of 25% HFCS and 20% sucrose

under multiple schedules of reinforcement. It was found that rats self-administered

much more sucrose than HFCS during IO SA. Following IO SA, mRNA for dopamine

receptor 2 and mu-opiod receptor genes were quantified in brain regions that are

18

implicated in addictive behaviours. Results indicated that although rats self-

administered much more sucrose than HFCS, only HFCS led to similar neural

adaptations that are present following repeated exposure to drugs of abuse. Thus, Levy

et al. (2015) concluded that HFCS may be a more potent reinforcer than sucrose in

terms of the behavioural and neural adaptations it produces following exposure.

Sweeteners (HFCS) in alcoholic beverages It has been suggested that the use of sweeteners in alcoholic beverages reduces

the aversive taste of alcohol, and leads to increased consumption in naïve drinkers

(Mosher & Johnsson, 2005; Lanier et al., 2005; Romanus, 2000; Copeland, Stevenson,

Gates, & Dillon, 2007). Indeed, youth that primarily consume sweetened alcoholic

beverages report a higher frequency of binge drinking episodes (Albers, Siegel,

Ramirez, Ross, DeJong, & Jernigan, 2015). Furthermore, it is well established that

animals consume greater quantities of alcohol when it is mixed in a sweet solution. In

fact, rats fail to acquire the self-administration of EtOH without the use of sucrose-

pairings, sucrose-fading or other lengthy initiation procedures (i.e. intermittent access

schedules, food/water deprivation; Samson, 1986; Samson, Pfeffer, & Tolliver, 1988).

Although this may seem intuitively obvious, the mechanisms by which

sweeteners increase alcohol intake are not fully understood. That is, sweeteners not

only change the taste of alcohol, but they also support reliable effects on behaviour and

the brain. Thus, sweeteners also have the potential to synergistically alter alcohol’s

reinforcing value. For example, in addition to increasing alcohol intake in animals, sweet

substances are also capable of eliciting approach behaviour (Yasoshima & Shimura,

2015), conditioning neutral stimuli (Sclafani & Ackroff, 2017; White & Carr, 1985; Agmo

19

& Marroquin, 1997), and maintaining high levels of operant self-administration (Levy et

al., 2015; Avena, Long, & Hoebel, 2005).

20

THE EFFECTS OF HIGH-FRUCTOSE CORN SYRUP ON ETHANOL TAKING, SEEKING AND PALATABILITY IN MALE RATS. Samantha Ayoub, Meenu Minhas, Erin Rock, Linda Parker and Francesco Leri Department of Psychology and Collaborative Neuroscience, University of Guelph, Ontario, Canada, N1G 2W1 Corresponding author:

Francesco Leri, PhD

Professor

Department of Psychology

University of Guelph

Guelph, Ontario N1G 2W1

Canada

E-mail: fleri@uoguelph.ca

519-824-4120 Ext: 58264

Acknowledgements

This research was funded by grants from the Natural Sciences and Engineering

Research Council (NSERC).

21

Abstract

Many alcoholic beverages contain sweeteners, such as high-fructose corn syrup

(HFCS), which has been implicated in increasing alcohol consumption and encouraging

hazardous drinking patterns. Therefore, four experiments were designed to investigate

whether the addition of HFCS to an ethanol (EtOH) solution could alter its intake,

hedonic taste, and sensitivity to food deprivation stress in Sprague Dawley rats.

Experiments 1 and 2 revealed that HFCS caused a robust increase in EtOH intake

during operant intraoral self-administration, however an equally palatable, yet non-

caloric sweetener (saccharin) could not replicate this effect. This suggests the caloric

value of a sweetener mediates the effect of sweeteners on EtOH intake. Experiments 3

and 4 revealed food deprivation stress increased EtOH consumption during resumption

following extinction, but only when HFCS was added to the solution. The palatability of

10% EtOH and 25% HFCS was also monitored across IO SA, and under conditions of

food deprivation stress. It was found that the palatability of 10% EtOH and 25% HFCS

increased following acquisition. More interestingly, food deprivation stress only

increased HFCS palatability. Thus, it could be the heightened palatability of HFCS

under stressful conditions that contributes to increased operant responding for solutions

containing HFCS during resumption in rats. These results have implications for the use

of highly palatable and caloric sweeteners in EtOH beverages because of the impact

they may have on excessive EtOH intake.

Key words

Operant self-administration, intraoral, alcohol intake, high-fructose corn syrup

22

Introduction Heavy episodic drinking refers to the consumption of 60 or more grams of alcohol

within a single occasion, occurring at least once per month (World Health Organization,

2014). This quantity of alcohol roughly equates to 5 standard drinks for men, and 4

standard drinks for women and is estimated to occur amongst 16% of drinkers

worldwide (World Health Organization, 2014). This statistic is concerning given that

heavy alcohol consumption is linked to numerous adverse health effects including, liver

and heart disease, birth defects, cancer, and unintentional injuries (Rehm et al., 2009).

In fact, the World Health Organization (2009) reported alcohol use as the third leading

risk factor contributing to the global burden of disease. In addition to the adverse health

effects associated with binge episodes (Massey & Arteel, 2012; Bala, Marcos, Gattu,

Catalano, & Szabo, 2014; Mathurin & Deltenre, 2009), the chronic consumption of large

quantities of alcohol increases an individual’s risk for subsequent disordered drinking

patterns and associated problem behaviours (Chassin, Pitts, & Prost, 2002; Hingson,

Heeren, & Winter, 2006).

Although there are several factors that facilitate the overconsumption of alcohol,

a key element appears to be the addition of sweeteners to alcoholic beverages (Mosher

& Johnsson, 2005; Copeland, Stevenson, Gates, & Dillon, 2007; Metzner & Kraus,

2008; Albers, Siegel, Ramirez, Ross, DeJong, & Jernigan, 2015). For example,

individuals that primarily consume sweetened alcoholic beverages have four times the

risk of experiencing alcohol-related injuries and report a higher frequency of heavy

episodic drinking (Albers et al., 2015). Furthermore, animal models show that

sweeteners increase the amount of alcohol self-administered in both home-cage and

23

operant self-administration procedures (Samson, Pfeffer, & Tolliver, 1988). In fact, most

species fail to acquire the self-administration of EtOH without the use of sucrose-fading

or other lengthy initiation procedures (i.e. intermittent access schedules, food/water

deprivation; Samson, Pfeffer, & Tolliver, 1988).

Sweet substances are known to have substantial and reliable effects on

behavior. For example, people describe the taste of sweeteners as pleasant (Thai et al.,

2011; Frank et al., 2008) and show preference for beverages that have been sweetened

(Tatzer, Schubert, Timischl, & Simbruner, 1985; Ventura & Mennella, 2011). In addition,

individuals display the motivation to consume sweetened beverages by exerting effort to

gain access to its administration (Schebendach et al., 2017; Guttman, 1954). The liking

and preference for sweeteners may be linked to activity in cortical regions that process

reward functions (Frank et al., 2008). Indeed, a variety of sweeteners increase the

release of dopamine into the nucleus accumbens (NAc; Rada, Avena & Hoebel, 2005;

Wheeler et al., 2011; Hajnal, Smith, & Norgren, 2004), a brain region implicated in

attributing incentive salience to rewarding stimuli (Wyvell & Berridge, 2000; Steinberg et

al., 2014). Although individuals show difficulty in discriminating between caloric and

non-caloric sweetness (Frank et al., 2008), caloric sweeteners are known to be more

potent reinforcers in terms of their behavioural and neurobiological effects (Domingos et

al., 2011; White & Carr, 1985). Some have even argued that caloric sweeteners may

have “addictive-like” properties (Levy et al., 2015; Lenoir, Serre, Cantin, Ahmed, 2007;

Madsen & Ahmed, 2014).

Recently, this laboratory has explored the behavioral and neurobiological

responses to high-fructose corn syrup in laboratory rats (HFCS; Levy et al., 2014; Levy

24

et al., 2015; Levy et al., 2018; Daniels, Marshall, & Leri, 2016). This sweetener was

selected for investigation because its consumption is highly prevalent in our diets

(Barlow, McKee, Basu, & Stuckler, 2017; Bray, 2013), and linked to several adverse

health effects including obesity, heart disease, and poor metabolic health (Meyers,

Mourra, & Beeler, 2017; Bray, 2013; Stanhope et al., 2015). In comparison to sucrose

(which contains 50% glucose and 50% fructose), HFCS has a higher proportion of

fructose (typically, 55% fructose), and this is sufficient to engender different behaviours

during operant intraoral self-administration in rats, as well as different patterns of gene

expression in the brain (Levy et al., 2015).

The overall aim of this study was to explore the impact of HFCS on operant

intraoral self-administration of ethanol (EtOH) in outbred male rats. This is a relevant

question because HFCS is used as sweetener in many alcoholic beverages available

for purchase (i.e. Smirnoff Ice, Mike’s Hard Lemonade, Bacardi Silver). It is generally

known that sweeteners change the taste of EtOH (Blednov et al., 2008) and facilitate

consumption in various species (Tolliver, Sadeghi, & Samson, 1988; Mandillo, Titchen,

& Miczek, 1998; Lanier, Hayes, & Duffy, 2004), but how HFCS interacts with EtOH’s

reinforcing effect is currently unknown.

Therefore, Experiment 1 was designed to establish whether HFCS can modify

operant intraoral self-administration (IO SA) of alcohol (EtOH; 5%, 10% and 20%

alc/vol) in outbred male rats. Experiment 2 was designed to contrast the effects of

HFCS and saccharin (SACC; a non-caloric sweetener) on EtOH IO SA. Experiment 3

tested the persistence of operant responding following extinction conditions in animals

trained to IO SA EtOH & HFCS. This experiment also included a stress manipulation,

25

which is known to alter the reinforcing effects of drugs of abuse and facilitate drug-

seeking in many species (Shaham, Erb, & Stewart, 2000). We selected food deprivation

stress (Shalev, Marinelli, Baumann, Piazza, & Shaham, 2003; Sinha, 2008) because of

the relevance to the behaviour explored (i.e., ingestion of calories from both EtOH and

HFCS). Finally, Experiment 4 expanded the findings of Experiment 3 by also

investigating how changes in EtOH and HFCS palatability, assessed by taste reactivity

(Grill & Norgren, 1978), may play a role in persistent drug-taking, drug-seeking and the

effect of food deprivation stress.

Methods

Subjects

Male Sprague Dawley rats were acquired at 175-200 grams (Charles River

Laboratories, Saint-Constant, Quebec) for all experiments. Animals were single-housed

and located in a colony room maintained on a 12-hour light/dark cycle (7:00/19:00h).

Standard rat chow and water were provided ad-libitum, unless indicated otherwise

below. Rats were acclimated to the colony room and handled daily for one week prior to

any experimental conditions. All procedures were reviewed and approved by The

University of Guelph Animal Care Committee and adhere to sanctions put forth by the

Canadian Council on Animal Care.

Apparatus

Self-administration chambers. Plexiglas operant conditioning chambers (model

ENV-008CT, Med Associates, Georgia, VT) were used for all SA procedures. Each

conditioning chamber was located within a larger sound- attenuating partition, with a

built-in fan to mask noise and provide constant ventilation. An overhead houselight (28-

26

V) was situated on the back wall of each chamber. On the front wall, a retractable lever

(8-cm above floor level) was situated just below a cue-light (28-V; 11-cm above floor

level). The retractable lever served as an active lever during SA sessions, and was

linked to a liquid infusion pump (Razel Scientific Instruments, Stamford, Connecticut).

On the opposite side of the front wall, there was a non-retractable lever (8-cm above

floor level) that served as an inactive lever during SA sessions. Stainless steel bars

provided the foundation of the conditioning chambers (30.5-cm x 24.1-cm x 21.0-cm). A

MED-PC interface controlled all conditioning chambers through MED-PC compatible

software.

Taste reactivity chambers. Taste reactivity chambers were constructed of clear

Plexiglas (22.5 x 26.0 x 20.0 cm) that sat atop a table with a clear glass top. A mirror

was placed beneath the chamber, tilted at a 45 angle to facilitate adequate viewing of

the rat’s ventral surface. Polyethylene tubing connected the IO cannula to an infusion

pump (KDS100; KD Scientific Inc., USA) which was set to deliver liquid for two minutes

at a rate of 0.5 ml/min. All taste reactivity sessions were recorded with a video camera

for experimenter scoring.

Surgery

IO cannulas were surgically implanted following a week of acclimation to the

colony room. The materials and procedures used for IO cannulation surgery have been

previously described in detail (Levy et al., 2014). Briefly, cannulas were constructed by

flanging polyethylene-90 tubing (approximately 80-mm in length) using a soldering iron.

Rats were then anesthetized using isoflurane, and cannulas were surgically implanted.

Using a 15-G stainless steel needle, cannulas were woven subcutaneously through the

27

skin at the back of the neck to protrude intraorally from the left cheek. A head-cap was

constructed using dental cement to uphold the bolt used to secure IO tubing during SA

sessions. Rats were given a week to recover following all surgical procedures.

General Procedures

Operant self-administration: Rats were food-restricted for all IO SA sessions by

allowing unlimited access to standard rat chow for only three hours daily. Feeding

occurred immediately following IO SA sessions. All IO SA sessions began 4-hrs into the

rat’s dark (active) cycle and ran for 180-min duration. Cannulas were flushed at the

beginning of every session with reverse osmosis water to ensure there were no

obstructions in the delivery line.

Rats were placed into the conditioning chambers and allowed to habituate for 5-

min prior to each session. Illumination of the house light signaled the beginning of a

session, with the active lever extending out after a 10-sec delay. Subsequently,

responding on the active lever resulted in a 0.1-ml IO infusion of liquid reinforcement on

a fixed-ratio 1 (FR1) or fixed-ratio 3 (FR3) schedule of reinforcement, and the

presentation of a cue-light (27.5-secs). During this time, responding on the active lever

was recorded but did not lead to additional reinforcement. Responding on the inactive

lever had no programmed consequences under any experimental conditions as it

served as a baseline of non-reinforced operant responding.

For the first three sessions of each IO SA experiment the lever-press response

was shaped by experimenter-induced priming. During priming sessions, rats were

allowed 15-min to perform an active lever-press response. If no response occurred

during this time, the experimenter held the rat up to the active lever and made the

28

animal press down to receive liquid reinforcement This was repeated every 15-min, up

to six times per session.

Extinction, cue-induced reinstatement and resumption: Once IO SA was stable

on a FR3 schedule of reinforcement (see below), rats received one 180-min extinction

session per day, for five days. Previous IO SA studies in our laboratory indicated that

after 5 days of extinction, responding on the active and inactive levers is comparable.

To maintain the oral sensation of liquid delivery during extinction, rats responded on the

active lever for a 0.1-ml IO delivery of reverse osmosis water. There was no

presentation of a cue-light during IO water delivery.

Cue-reinstatement testing was conducted during a 180-minute session following

the last day of extinction. During reinstatement, rats could respond on the active lever

for a 0.1 ml IO delivery of reverse osmosis water and the illumination of the cue light.

Resumption tests were conducted in an identical manner, but rats could respond

on the active lever for a 0.1 ml IO delivery of EtOH, HFCS or EtOH + HFCS and the

presentation of a cue-light.

Extinction, reinstatement and resumption sessions were tested on a FR3

schedule of reinforcement. Active and inactive lever responding, and number of

infusions earned were recorded.

Experimental procedures

Experiment 1: IO SA of EtOH and combined EtOH + HFCS.

Rats (n=8) self-administered 5% EtOH on a FR1 schedule of reinforcement for

16 consecutive 180-min sessions. The response requirement was increased to a FR3

schedule of reinforcement for the next four sessions (17-20), and then reduced back to

29

an FR1 schedule for four sessions (21-24). This alternation in schedule was performed

to ascertain baseline level of EtOH self-administration. On session 25, 5% EtOH was

mixed in a 25% HFCS solution and animals self-administered the mixed solution on a

FR1 schedule. The FR3 schedule was then reintroduced for 8 additional sessions (26-

33). Following this period, the concentration of EtOH in 25% HFCS was varied in blocks

of four sessions to complete a dose-response curve. That is, rats self-administered a

10% EtOH-HFCS solution for four sessions (34 – 37), then a 20% EtOH-HFCS solution

for four sessions (38 – 41), and then a 5% EtOH-HFCS solution for another four

sessions (42 – 45). To conclude the experiment, HFCS was removed and rats reverted

to self-administering 5% EtOH for four sessions (45-49).

Experiment 2: IO SA of EtOH and combined EtOH + SACC

Rats (n=14) self-administered a 5% EtOH solution mixed in 0.1% saccharin for

10 180-min sessions. Next, the schedule of reinforcement was increased to a FR3

schedule for seven more sessions (11-17). Subsequently, a dose-response curve was

performed as in Experiment 1: the concentration of EtOH was successively increased

(5%, 10%, 20%, 5%) in the 0.1% saccharin solution (sessions 17-32). Following the

dose-response determination, rats received a single session (33) in which they could

respond for 25% HFCS. This session tested whether this group of rats could emit more

responses for a more effective reinforcer (Levy et al. 2016).

Experiment 3: Effect of food deprivation stress on IO SA acquisition, extinction and

resumption of EtOH + HFCS

Rats (n=28) self-administered 10% EtOH in 25% HFCS on an FR1 schedule for

10 180-min sessions (1-10), and then the schedule of reinforcement was increased to

30

an FR3 schedule for 7 sessions (11-17). The concentration of EtOH was selected based

on the results of Experiments 1-2 which determined 10% EtOH + 25% HFCS led to the

highest g/kg alcohol intake (0.91 g/kg). Following 5 180-min extinction sessions, rats

were randomly assigned to food restriction (n=14) or food deprivation (n=14) conditions.

Food restricted rats continued their typical food restriction regimen of 3-hours of

free access to standard rat chow, and the test of cue-induced reinstatement was

followed by two tests of resumption 10% EtOH and 25% HFCS intake, which occurred

on two counterbalanced sessions.

Food deprived rats did not receive any food after their last extinction session, but

they were fed after the test of cue-induced reinstatement. To control for food deprivation

stress carry over effects, they received one more extinction session followed by EtOH -

only or HFCS-only resumption test, and they were fed immediately after the test. This

was repeated once more to counterbalance for the substance self-administered.

Experiment 4: Effect of food deprivation on resumption of EtOH + HFCS IO SA

responding-modulation by palatability of components.

A within-subjects design was used to investigate the effect of food deprivation

stress on 10% EtOH + 25% HFCS intake during IO SA resumption testing. Additionally,

a taste reactivity (TR) test was given at four different time points throughout the SA

period to assess possible changes in palatability of 10% EtOH and 25% HFCS under

pre-acquisition, post-acquisition, food restriction and food deprivation conditions. The

timeline of procedural events that occurred during Experiment 4 is represented in Figure

4A.

The day preceding the first TR test, rats were habituated to TR chambers by

31

intraorally infusing reverse osmosis water at a flow rate of 0.5 ml/min for two minutes.

During TR tests rats received 10% EtOH and 25% HFCS separately over a two-min

exposure period. The appropriate liquid was dispensed at the same flow rate of 0.5

ml/min, resulting in 1 ml of the appropriate solution dispensed over a two-min period.

EtOH and HFCS solutions were presented to rats an hour following an IO SA session

and these solutions were tested in a counterbalanced order across rats. All test

sessions were captured on video and tapes were scored for the mean number of

hedonic reactions of tongue protrusions (protrusions of the tongue from the mouth) and

mouth movements (summed). Additionally, tapes were scored for the mean number of

aversive reactions of gaping (wide triangular shaped open mouth exposing lower

incisors) and the avoidance reaction of passive dripping (passive dripping of liquid from

the mouth to the floor). The aversive reactions of chin rubbing (rubbing chin along the

floor of the cage) and paw treading (movement of forepaws back and forth) were also

assessed, but these behaviors were not seen so they are not discussed further.

Rats (n=18) self-administered 10% EtOH + 25% HFCS on a FR1 schedule of

reinforcement for 10 consecutive 180-min sessions. The response requirement was

increased to a FR3 schedule of reinforcement for the next seven sessions (11-17). On

session 25, rats received five extinction sessions (18-22), followed by tests of cue-

induced reinstatement and resumption of 10% EtOH + 25% HFCS IO SA in both a food

deprived and food restricted state. Extinction sessions were interposed between each

test day as described above.

Drugs

Ethanol solutions (5%, 10%, 20%) were prepared by diluting ethyl alcohol (95%

32

vol; Commercial Alcohols, Tiverton, Ontario) in reverse osmosis water. High-fructose

corn syrup (25%; HFCS-55 formula; Natures Flavors, CA, USA) and saccharin (0.1%;

sodium salt hydrate 99+ %, ACROS Organics, New Jersey) solutions were also

prepared by dilution in reverse osmosis water. Solutions were freshly prepared every

three days to prevent evaporation.

Statistical analyses

One factor analyses of variance (ANOVA) and t-tests with independent or

repeated subjects were conducted as appropriate. Significant main effects or

interactions were further analysed by multiple comparisons using the Student-Newman

Keuls method (α=0.05). All analyses were performed using SigmaStat (v. 12.5).

Results

Experiment 1: IO SA of EtOH and combined EtOH + HFCS.

Rats demonstrated higher IO SA of 5% EtOH + 25% HFCS than 5% EtOH

alone. Figure 1A represents the mean (SEM) infusions rats earned across 25 days of IO

SA acquisition for 5% EtOH (sessions 1-24) and 5% EtOH + 25% HFCS (session 25).

An ANOVA revealed a main effect of session [F(7, 24) = 20.934, p < 001]. Multiple

comparisons indicated that the number of infusions rats earned on session 25 was

significantly higher than sessions 1 – 24, p < 0.001. No other significant differences

were found.

In a dose-response assessment, when 20% EtOH was added to HFCS, rats

showed suppressed IO SA. Figure 1B represents the mean (SEM) infusions rats earned

across sessions 26 – 45, during which the concentration of EtOH in 25% HFCS was

varied to create a dose-response (intake) curve. Blocks of four sessions were used to

33

allow IO SA behaviour to stabilize and the last session of which each concentration was

presented were compared (sessions 33, 37, 41 and 45). An ANOVA revealed a main

effect of session [F (3, 15) = 19.872, p < 0.001]. Multiple comparisons indicated rats

earned significantly less infusions on session 41 (20% EtOH) than on sessions 33 (5%

EtOH; p < 0.001), 37 (10% EtOH; p < 0.001) and 45 (5% EtOH; p < 0.001). No other

significant differences were found.

Rats displayed enhanced IO SA of 5% EtOH following training with 5% EtOH +

25% HFCS. Figure 1C represents the mean (SEM) infusions rats earned on the last day

of acquisition for 5% EtOH prior to HFCS pairing (FR3; session 20) compared to the

four sessions after HFCS was removed (FR3; sessions 46-49). An ANOVA revealed a

significant effect of session [F (4, 16) = 4.675, p = 0.11]. Multiple comparisons indicated

rats consumed less 5% EtOH on session 20 than sessions 46 (p < 0.01), 47 (p < 0.05),

and 49 (p < 0.05). No other significant differences were found.

Experiment 2. IO SA of EtOH and combined EtOH + SACC.

Saccharin was much less effective than HFCS in enhancing IO SA of EtOH .

Figure 2A represents the mean (SEM) infusions rats earned across 16 days of IO SA

acquisition for 5% EtOH in 0.1% SACC. An ANOVA revealed a main effect of session [F

(13, 208) = 2.038, p = 0.012]. Multiple comparisons indicated that infusions earned by

rats did not differ between session 1 and session 17. In other words, although infusions

earned varied across sessions, there was not a significant increase in 5% EtOH in 0.1%

SACC intake between the first and last day of IO SA. Figure 2B represents the mean

(SEM) number of infusions rats earned on the last session of Experiment 1 (session 49;

FR3; 5% EtOH) and the last session of Experiment 2 (session 30; FR3; 5% EtOH in

34

0.1% SACC). An independent samples t-test revealed rats earned more infusions when

EtOH was mixed in 0.1% SACC, [t(20)= -2.68, p=0.01]. Figure 2C represents the mean

(SEM) number of infusions rats earned across sessions 14 to 29 during which the

concentration of EtOH in 0.1% SACC was varied to create a dose-response (intake)

curve. Blocks of four sessions were used to allow IO SA behaviour to stabilize and the

last session of which each concentration was presented were compared (sessions 17,

21, 25, and 29). An ANOVA revealed main effect of session [F (3, 39) = 3.741, p <

0.019]. Multiple comparisons indicated rats earned significantly fewer infusions on

session 25 (20% EtOH) than on session 17 (5% EtOH; p < 0.001). No other significant

differences were found. Figure 2D represents the mean (SEM) number of infusions rats

earned during the IO SA dose-response (intake) curve for EtOH in SACC (Experiment

2) and EtOH in HFCS (Experiment 1). An ANOVA revealed a solution by concentration

interaction [F (3, 72) = 8.324, p < 0.001], a main effect of solution [F (1, 72) = 127.387, p

< 0.001] and a main effect of concentration [F (3, 72) = 16.392, p < 0.001]. Multiple

comparisons on marginal means indicated that higher concentrations of EtOH

significantly reduced intake and that animals consumed significantly more solution when

HFCS was added to EtOH than when SACC was added.

Experiment 3. Effect of food deprivation stress on cue-induced and EtOH or HFCS-

induced resumption of responding.

Rats displayed enhanced responding for EtOH + HFCS across acquistion

sessions. Following extinction, food deprivation stress enhanced cue-induced and

HFCS (but not EtOH)-induced resumption of responding. Figure 3A represents the

mean (SEM) infusions rats earned across 17 days of IO SA of 10% EtOH in 25% HFCS.

35

An ANOVA across sessions revealed a main effect of session [F (1, 27) = 4.690, p <

0.05]. Multiple comparisons indicated rats significantly increased their intake from FR1-1

to FR1-10 (p < 0.001) and from FR3-1 to FR3-7. Furthermore, the number of infusions

earned did not significantly differ across the last four sessions of each schedule of

reinforcement. Figure 3B represents the mean (SEM) active lever responses rats made

on the last day of IO SA acquisition (Baseline; B) and the following 5 extinction

sessions. An ANOVA revealed a main effect of session [F (5, 162) = 100.178, p <

0.001]. Multiple comparisons indicated response rates were lower across all extinction

sessions when compared to the baseline responding during IO SA acquisition.

Furthermore, extinction sessions 2 – 5 produced significantly lower lever responding

when compared to the first extinction session. No other significant differences were

found. Figure 3C represents the mean (SEM) active lever responses made on the test

of cue reinstatement in food restricted and food deprived rats. A t-test revealed food

restricted rats had lower levels of responding compared to food deprived rats when

tested for cue-induced reinstatement, [t(26)= -2.166, p < 0.05]. Figure 3D represents the

mean (SEM) infusions rats earned on the tests of EtOH and HFCS resumption.

Independent samples t-tests revealed food deprivation stress increased HFCS intake

during resumption, [t(26)= 4.068, p=0.001] but had no effect on EtOH intake [t(26)=

0.148, p=0.884].

Experiment 4: Effect of food deprivation on resumption of EtOH + HFCS IO SA

responding-modulation by palatability of components.

Food deprivation stress enhanced EtOH+HFCS resumption of responding

following extinction. Both EtOH and HFCS individually elicited enhanced hedonic

36

responses in the TR test following acquisition training, and food deprivation stress

during resumption testing enhanced hedonic responses of HFCS, but not EtOH. Figure

4B represents the mean (SEM) active lever responses made during EtOH + HFCS

resumption following extinction across food restricted and food deprived conditions. A

paired samples t-test revealed that food deprived rats consumed more EtOH + HFCS

during resumption than rats under food restriction conditions [t(17)= -2.213, p < 0.05].

Figure 4C represents the mean (SEM) hedonic taste responses elicited by EtOH or

HFCS during baseline testing compared to post-acquisition taste reactivity testing. A

paired samples t-test revealed hedonic taste reactions were increased during post-

acquisition compared to baseline TR testing for EtOH [t(19)= -5.098, p < 0.001] and

HFCS [t(19)= -2.311, p < 0.05]. There was no effect on aversive taste reactions to EtOH

and HFCS (data not shown). Figure 4D represents the mean (SEM) hedonic taste

responses elicited by EtOH or HFCS during taste reactivity testing for food restricted

and food deprived rats. Paired samples t-tests revealed that food deprivation stress

increased HFCS palatability [t(19) = -3.733, p < 0.001] but not EtOH palatability [t(19)=

1.027, p =0.318].

Discussion

Young, inexperienced and often under-aged drinkers report a high frequency of

heavy episodic drinking patterns (Archie, Kazemi, & Akhtar-Danesh, 2012) and adding

sweeteners to alcohol may increase the risk of problematic drinking in this population.

Therefore, we designed four experiments in naïve Sprague Dawley rats to investigate

whether the addition of a caloric sweetener (HFCS) to an EtOH solution could alter its

intake, hedonic taste, and sensitivity (intake under) to food deprivation stress. In

37

Experiment 1 (to study the effect of HFCS on EtOH intake) rats were trained to

intraorally self-administer 5% EtOH under multiple schedules of reinforcement. The

addition of 25% HFCS caused a robust increase in EtOH intake during IO SA.

Additionally, when HFCS was removed and rats reverted to self-administering the 5%

EtOH solution, their intake was elevated from levels seen before the HFCS pairings.

Using similar methods, Experiment 2 revealed an equally palatable, yet non-caloric

sweetener (SACC) did not increase EtOH intake to the degree that was observed with

HFCS. In Experiments 3 and 4 (to study the effect of HFCS on EtOH’s sensitivity to food

deprivation stress) rats were trained to self-administer 10% EtOH in 25% HFCS. This

concentration of EtOH was chosen based on results from Experiment 1. Following

acquisition, rats underwent behavioural extinction to test the persistence of operant

responding during cue-induced reinstatement and resumption (of 10% EtOH, 25%

HFCS, and 10% EtOH + 25% HFCS) under food deprivation stress. Food deprivation

stress increased 25% HFCS and 10% EtOH + 25% HFCS intake, but did not alter the

intake of 10% EtOH during resumption. Finally, the changes in palatability of 10% EtOH

and 25% HFCS were assessed using taste reactivity at multiple time points throughout

the IO SA of 10% EtOH + 25% HFCS. Rats elicited more hedonic orofacial responses to

10% EtOH and 25% HFCS following IO SA of 10% EtOH + 25% HFCS. Furthermore,

food deprivation stress increased hedonic orofacial responses to the 25% HFCS

solution, but did not alter 10% EtOH palatability. Thus, it could be the increased

palatability of HFCS under food deprivation conditions that contributes to the increased

motivation to consume HFCS during operant IO SA resumption in rats. These results

38

have implications for the use of highly palatable and caloric sweeteners in EtOH

beverages because of the impact they may have on excessive EtOH intake.

When rats could self-administer 5% EtOH during IO SA, intake was minimal.

However, when the same concentration of EtOH was sweetened with 25% HFCS, a

robust increase in EtOH intake was observed (see Figure 1A). Following this, a dose-

response (intake) curve was produced by varying the concentration of EtOH within

HFCS. Rats maintained the number of infusions earned when the concentration of

EtOH was increased from 5% to 10%, however a further increase to 20% EtOH reduced

the amount of solution consumed (see Figure 1B.). In other words, the 10% EtOH +

25% HFCS solution produced the highest levels of EtOH intake during IO SA. Finally,

when rats reverted to self-administering 5% EtOH, their intake was elevated from levels

seen before the HFCS pairings (see Figure 1C.). It is important to note that although

SACC increased the intake of EtOH during IO SA, this effect was miniscule in

comparison to what was observed when HFCS was used to sweeten the EtOH solution

(see Figure 2).

Caloric sweeteners such as sucrose and glucose increase EtOH intake during

SA in rodents (Samson, Pfeffer, & Tolliver, 1988); however, to our knowledge, we are

the first to demonstrate this effect using HFCS. HFCS was selected as its use is rapidly

increasing throughout North America, yet its effects on behaviour and the brain are still

not well understood. Additionally, HFCS is found in many of the alcoholic beverages

available for purchase (i.e. Bacardi Silver, Palm Bay, Mike’s Hard Lemonade). In

addition to increasing EtOH intake, caloric sweeteners are also capable of eliciting

approach behaviour (Yasoshima & Shimura, 2015) conditioning neutral stimuli (Sclafani

39

& Ackroff, 2017; White & Carr, 1985; Agmo & Marroquin, 1997), and maintaining high

levels of operant self-administration (Levy et al., 2015; Avena, Long, & Hoebel, 2005).

That is, caloric sweeteners are known to have strong behavioural reinforcing properties.

Therefore, it is not surprising that rats increased their EtOH intake when HFCS was

mixed into the EtOH solution. We suggest this increase was mostly caused by caloric

reinforcement, because an equally palatable yet non-caloric sweetener did not cause

such a drastic increase in IO SA behaviour. Although non-caloric sweeteners hold

similar hedonic taste properties as do caloric sweeteners (Frank et al., 2008), it is

known that their behavioural reinforcing properties are minimal in comparison

(Domingos et al., 2011; White & Carr, 1985).

Interestingly, our study also suggests that HFCS may have indirect effects on

EtOH intake. That is, once HFCS was removed from EtOH following multiple pairings,

EtOH intake was elevated in comparison to initial IO SA levels. While we understand

that many factors could have contributed to this effect (increased palatability of EtOH,

tolerance) it could be that HFCS conditioned or altered EtOH’s reinforcing value. After

all, caloric sweeteners are known to modify the reinforcing value of neutral stimuli

(Sclafani & Ackroff, 2017; Sclafani, Glass, Margolskee, & Glendinning, 2010; Fedorchak

& Bolles, 1987; White & Carr, 1985; Agmo & Marroquin, 1997; Delamater, Sclafani, &

Bodnar, 2000). To test this, future research could employ methods that are more

suitable for measuring the reinforcing value of stimuli, such as, progressive ratio operant

testing or choice procedures (Reilly, 1999; Sclafani & Ackroff, 2003; Butler, Irons,

Bassett, & Correia, 2018).

40

Once the effect of HFCS on EtOH consumption was determined, our next

objective was to assess how HFCS could alter EtOH’s sensitivity to food deprivation

stress. Food deprivation stress has been shown to elevate corticosterone levels in rats

(McGhee, Jefferson, & Kimball, 2009) and facilitate the reinstatement of operant self-

administration for drugs of abuse (Shalev, Marinelli, Baumann, Piazza, & Shaham 2003;

Highfield et al, 2002; Mantsch, Baker, Funk, Le & Shaham, 2016). Food deprivation

stress was also chosen based on its relevance to the behaviour being explored (i.e.,

ingestion of caloric substances). First, rats acquired IO SA of 10% EtOH + 25% HFCS

(see Figure 3A) then this behaviour was extinguished (see Figure 3B). The absence of

the cue-light during extinction conditions is important to establish that this stimulus only

represents sweetened EtOH reinforcement. The use of water during extinction

conditions is also crucial to maintain the orosensory stimulation that is experienced

during IO SA acquisition. During cue-induced reinstatement food deprivation stress

increased responding for water reinforcement (see Figure 3C). During resumption

testing, food deprivation stress did not increase 10% EtOH intake, however it increased

the intake of 25% HFCS. Finally, Experiment 4 revealed that food deprivation stress

increased the intake of 10% EtOH + 25% HFCS (see Figure 4A) and the palatability of

25% HFCS, but not 10% EtOH.

In this study, the lack of an effect of food deprivation stress on EtOH intake

during resumption was surprising. This is because stress has long been implicated in

exacerbating relapse-like responding for EtOH in humans. For example, the experience

of a stressor in abstinent alcoholics is associated with increased EtOH cravings (Clay et

al., 2018; Fox, Bergquist, Hong, & Sinha, 2007). Additionally, the occurrence of severe

41

stress following treatment is correlated with increased relapse in recovering alcoholics

(Brown, Vik, Patterson, Grant, & Schuckit, 1995; Brown et al., 1990). Finally, exposure

to acute stress increases alcohol consumption in heavy drinkers (Thomas, Bacon,

Randall, Brady, & Ronald, 2011; McGrath, Jones, & Field, 2016). In rodents however,

the effect of stress on EtOH consumption is inconsistent (Pohorecky, 1990) with some

researchers reporting decreases (Sprague & Maickel, 1994; Vengeliene, Siegmund,

Singer, Sinclair, Li, & Spanagel, 2003) while others report increases (Bahi, 2013;

Gomez, Lewis, & Luine, 2012; Vengeliene, et al., 2003). Interestingly, food deprivation

stress increased EtOH intake only when HFCS was mixed in the solution. These results

imply that HFCS increases EtOH’s sensitivity to the effect of food deprivation stress.

Thus, HFCS alters EtOH intake under normal and stressful conditions and this should

be considered when employing efforts to reduce excessive EtOH intake.

The last objective of this study was to assess how the palatability of 10% EtOH

and 25% HFCS is altered throughout the IO SA of 10% EtOH in 25% HFCS. More

specifically, taste reactivity was used to test the palatability of each solution pre-

acquisition (naïve responding), post-acquisition (experienced responding) and under

conditions of food deprivation stress (see Figure 4A). The number of palatable orofacial

responses to EtOH and HFCS increased following IO SA of 10% EtOH in 25% HFCS

(see Figure 4 C.). Interestingly, the number of aversive orofacial responses (i.e. gaping)

did not change for either solution following IO SA experience (data not shown). While

rats displayed no aversive responses to the taste of 25% HFCS, gaping occurred during

pre- and post-acquisition for the 10% EtOH solution (data not shown). During

42

resumption, food deprivation stress increased palatable orofacial responses to 25%

HFCS, but did not alter EtOH palatability (see Figure 4D).

This data suggests that the IO SA of 10% EtOH in 25% HFCS alters the hedonic

palatability of EtOH, without altering its aversive taste properties. In contrast, Kiefer,

Bice, and Badia-Elder (1994) have shown no change in hedonic taste, but a decrease in

aversive taste responses following continuous home-cage access to a variety of EtOH

concentrations. The lack of difference between aversive taste responses during pre-

and post-acquisition may be explained by the use of 25% HFCS during EtOH exposure.

That is, our rats never experienced EtOH by itself during the exposure period as it was

always mixed in 25% HFCS. The effect of food deprivation on EtOH and HFCS

palatability may be the most interesting finding from the taste reactivity data because it

corroborates with the self-administration results from Experiments 3 and 4. Food

deprivation stress had no effect on EtOH intake under resumption testing, nor did it

affect palatability. On the other hand, food deprivation stress significantly increased

HFCS intake under resumption and increased its palatability. We suggest that the

increased palatability of HFCS under stressful conditions may contribute to the

increased motivation to consume HFCS during operant responding.

These data imply the use of HFCS in alcoholic beverages encourages EtOH

consumption, increases its sensitivity to food deprivation stress, and increases its

hedonic taste. Efforts aimed at reducing the harmful consumption of EtOH in naïve

drinkers should include removal of HFCS from the list of ingredients.

43

References

Agmo, A., & Marroquin, E. (1997). Role of gustatory and postingestive actions of

sweeteners in the generation of positive affect as evaluation by place preference

conditioning. Appetite, 29(3), 269-289.

Albers, A. B., Siegel, M., Ramirez, R. L., Ross, C., DeJong, W., & Jernigan, D. H.

(2015). Flavored alcoholic beverage use, risky drinking behaviors, and adverse

outcomes among underage drinkers: Results from the ABRAND study. American

Journal of Public Health, 105(4), 810-815.

Anderson, P., de Bruijn, A., Angus, K., Gordon, R., & Hastings, G. (2009). Impact of

alcohol advertising and media exposure on adolescent alcohol use: A systematic

review of longitudinal studies. Alcohol and Alcoholism, 44(3), 229-243.

Archie, S., Zangeneh Kazemi, A., & Akhtar-Danesh, N. (2012). Concurrent binge

drinking and depression among canadian youth: Prevalence, patterns, and

suicidality. Alcohol, 46(2), 165-72.

Avena, N. M., Long, K. A., & Hoebel, B. G. (2005). Sugar-dependent rats show

enhanced responding for sugar after abstinence: Evidence of a sugar deprivation

effect. Physiology & Behavior, 84(3), 359-362.

Bahi, A. (2013). Increased anxiety, voluntary alcohol consumption and ethanol-induced

place preference in mice following chronic psychosocial stress. Stress, 16(4),

441-451.

Bala, S., Marcos, M., Gattu, A., Catalano, D., & Szabo, G. (2014). Acute binge drinking

increases serum endotoxin and bacterial DNA levels in healthy individuals. PLoS

One, 9(5).

44

Barlow, P., McKee, Basu, & Stuckler, (2017). Impact of the north american free trade

agreement on high-fructose corn syrup supply in canada: A natural experiment

using synthetic control methods. Canadian Medical Association Journal, 189(26),

E881-E887.

Blednov, Y. A., Walker, D., Martinez, M., Levine, M., Damak, S., & Margolskee, R. F.

(2008). Perception of sweet taste is important for voluntary alcohol consumption

in mice. Genes, Brain, and Behavior, 7(1), 1-13.

Bray, G. A. (2013). Energy and fructose from beverages sweetened with sugar or high-

fructose corn syrup pose a health risk for some people. Advances in

Nutrition, 4(2), 220-225.

Brown, S. A., Vik, P. W., McQuaid, J. R., Patterson, T. L., Irwin, M. R., & Grant, I.

(1990). Severity of psychosocial stress and outcome of alcoholism treatment. Journal

of Abnormal Psychology, 99(4), 344-348.

Brown, S. A., Vik, P. W., Patterson, T. L., Grant, I., & Schuckit, M. A. (1995). Stress,

vulnerability and adult alcohol relapse. Journal of Studies on Alcohol, 56(5), 538-

545.

Butler, L. H., Irons, J. G., Bassett, D. T., & Correia, C. J. (2018). Using the multiple-

choice procedure to measure the relative reinforcing efficacy of gambling: Initial

validity evidence among college students. Journal of Gambling Studies, 34(2),

513-520.

Chassin, L., Pitts, S. C., & Prost, J. (2002). Binge drinking trajectories from adolescence

to emerging adulthood in a high-risk sample: Predictors and substance abuse

outcomes. Journal of Consulting and Clinical Psychology, 70(1), 67-78.

45

Clay, J. M., Adams, C., Archer, P., English, M., Hyde, A., Stafford, L. D., & Parker, M.

O. (2018). Psychosocial stress increases craving for alcohol in social drinkers:

Effects of risk-taking. Drug and Alcohol Dependence, 185, 192-197.

Copeland, J., Stevenson, R. J., Gates, P., & Dillon, P. (2007). Young australians and

alcohol: The acceptabllity of ready-to-drink (RTD) alcoholic beverages among 12-

30-year-olds. Addiction, 102(11), 1740-1746.

Daniels, S., Marshall, P., & Leri, F. (2016). Alterations of naltrexone-induced

conditioned place avoidance by pre-exposure to high fructose corn syrup or

heroin in sprague-dawley rats. Psychopharmacology, 233(3), 425-433.

Delamater, A. R., Sclafani, A., & Bodnar, R. J. (2000). Pharmacology of sucrose-

reinforced place-preference conditioning: Effects of naltrexone. Pharmacology

Biochemistry and Behavior, 65(4), 697-704

Domingos, A. I., Vaynshteyn, J., Voss, H. U., Ren, X., Gradinaru, V., Zang, F., . . .

Friedman, J. (2011). Leptin regulates the reward value of nutrient. Nature

Neuroscience, 14(12), 1562-8.

Fedorchak, P. M., & Bolles, R. C. (1987). Hunger enhances the expression of calorie-

but not taste-mediated conditioned flavor preferences. Journal of Experimental

Psychology.Animal Behavior Processes, 13(1), 73-79.

Frank, G. K. W., Oberndorfer, T. A., Simmons, A. N., Paulus, M. P., Fudge, J. L., Yang,

T. T., & Kaye, W. H. (2008). Sucrose activates human taste pathways differently

from artificial sweetener. NeuroImage, 39(4), 1559-1569.

Fox, H. C., Bergquist, K. L., Hong, K., & Sinha, R. (2007). Stress-induced and alcohol

cue-induced craving in recently abstinent alcohol-dependent individuals. Alcoholism,

46

Clinical and Experimental Research, 31(3), 395-403.

Gomez, J. L., Lewis, M. J., & Luine, V. N. (2012). The interaction of chronic restraint

stress and voluntary alcohol intake: Effects on spatial memory in male rats.

Alcohol, 46(5), 499-504.

Grill, H. J., & Norgren, R. (1978). The taste reactivity test. I. mimetic responses to

gustatory stimuli in neurologically normal rats. Brain Research, 143(2), 263-279.

Guttman, N. (1954). Equal-reinforcement values for sucrose and glucose solutions

compared with equal-sweetness values. Journal of Comparative and

Physiological Psychology, 47(5), 358-361.

Hajnal, A., Smith, G. P., & Norgren, R. (2004). Oral sucrose stimulation increases

accumbens dopamine in the rat. American Journal of Physiology. Regulatory,

Integrative and Comparative Physiology, 286(1), R31-R37.

Highfield, D. A., Mead, A. N., Grimm, J. W., Rocha, B. A., & Shaham, Y. (2002).

Reinstatement of cocaine seeking in 129X1/SvJ mice: Effects of cocaine priming,

cocaine cues and food deprivation. Psychopharmacology, 161(4), 417-424.

Hingson, R. W., Heeren, T., & Winter, M. R. (2006). Age of alcohol-dependence onset:

Associations with severity of dependence and seeking treatment.

Pediatrics, 118(3), e755-e763.

Kiefer, S. W., Bice, P. J., & Badia-Elder, N. (1994). Alterations in taste reactivity to

alcohol in rats given continuous alcohol access followed by abstinence.

Alcoholism, Clinical and Experimental Research, 18(3), 555-559.

47

Lanier, S. A., Hayes, J. E., & Duffy, V. B. (2005). Sweet and bitter tastes of alcoholic

beverages mediate alcohol intake in of-age undergraduates. Physiology &

Behavior, 83(5), 821-831.

Lenoir, M., Serre, F., Cantin, L., & Ahmed, S. H. (2007). Intense sweetness surpasses

cocaine reward. PloS One, 2(8), 1.

Levy, A., Limebeer, C. L., Ferdinand, J., Shillingford, U., Parker, L. A., & Leri, F. (2014).

A novel procedure for evaluating the reinforcing properties of tastants in

laboratory rats: Operant intraoral self-administration. Journal of Visualized

Experiments, 84, 1.

Levy, A., Daniels, S., Hudson, R., Horman, T., Flynn, A., Zhou, Y., & Leri, F. (2018).

Bupropion and naltrexone combination alters high fructose corn syrup self-

administration and gene expression in rats. Neuropharmacology, 135, 547-554.

Levy, A., Marshall, P., Zhou, Y., Kreek, M. J., Kent, K., Daniels, S., . . . Leri, F. (2015).

Fructose: Glucose ratios-A study of sugar self-administration and associated

neural and physiological responses in the rat. Nutrients, 7(5), 3869-3890.

Madsen, H. B., & Ahmed, S. H. (2015). Drug versus sweet reward: Greater attraction to

and preference for sweet versus drug cues. Addiction Biology, 20(3), 433-444.

Mandillo, S., Titchen, K., & Miczek, K. A. (1998). Ethanol drinking in socially housed

squirrel monkeys. Behavioural Pharmacology, 9(4), 363-367.

Mathurin, P., & Deltenre, P. (2009). Effect of binge drinking on the liver: An alarming

public health issue? Gut, 58(5), 613-617.

48

Mantsch, J. R., Baker, D. A., Funk, D., Lê, A.,D., & Shaham, Y. (2016). Stress-induced

reinstatement of drug seeking: 20 years of progress.

Neuropsychopharmacology, 41(1), 335-356.

Massey, V. L., & Arteel, G. E. (2012). Acute alcohol-induced liver injury. Frontiers in

Physiology, 3, 193.

McGhee, N. K., Jefferson, L. S., & Kimball, S. R. (2009). Elevated corticosterone

associated with food deprivation upregulates expression in rat skeletal muscle of

the mTORC1 repressor, REDD1. Journal of Nutrition, 139(5), 828-834.

McGrath, E., Jones, A., & Field, M. (2016). Acute stress increases ad-libitum alcohol

consumption in heavy drinkers, but not through impaired inhibitory

control.Psychopharmacology, 233(7), 1227-1234.

Metzner, C., & Kraus, L. (2008). The impact of alcopops on adolescent drinking: A

literature review. Alcohol and Alcoholism, 43(2), 230-239.

Meyers, A. M., Mourra, D., & Beeler, J. A. (2017). High fructose corn syrup induces

metabolic dysregulation and altered dopamine signaling in the absence of

obesity. PLoS One, 12(12).

Mosher, J. F., & Johnsson, D. (2005). Flavored alcoholic beverages: An international

marketing campaign that targets youth. Journal of Public Health Policy, 26(3),

326-342.

Pohorecky, L. A. (1990). Interaction of ethanol and stress: Research with experimental

animals -- an update. Alcohol Alcoholism., 25(2-3), 263-276.

Rada, P., Avena, N. M., & Hoebel, B. G. (2005). Daily bingeing on sugar repeatedly

releases dopamine in the accumbens shell. Neuroscience, 134(3), 737-744.

49

Reilly, S. (1999). Reinforcement value of gustatory stimuli determined by progressive

ratio performance. Pharmacology, Biochemistry, and Behavior, 63(2), 301-311.

Samson, H. H., Pfeffer, A. O., & Tolliver, G. A. (1988). Oral ethanol self-administration

in rats: Models of alcohol-seeking behavior. Alcoholism, Clinical and

Experimental Research, 12(5), 591-598.

Sclafani, A., & Ackroff, K. (2017). Flavor preferences conditioned by nutritive and non-

nutritive sweeteners in mice. Physiology & Behavior, 173, 188-199.

Sclafani, A., & Ackroff, K. (2003). Reinforcement value of sucrose measured by

progressive ratio operant licking in the rat. Physiology & Behavior, 79(4-5), 663-

670.

Sclafani, A., Bodnar, R. J., & Delamater, A. R. (1998). Pharmacology of food

conditioned preferences. Appetite, 31(3), 406-p. 406.

Sclafani, A., Glass, D. S., Margolskee, R. F., & Glendinning, J. I. (2010). Gut T1R3

sweet taste receptors do not mediate sucrose-conditioned flavor preferences in

mice. American Journal of Physiology. Regulatory, Integrative and Comparative

Physiology, 299(6), R1643-R1650.

Schebendach, J., Klein, D. A., Mayer, L. E. S., Attia, E., Devlin, M. J., Foltin, R. W., &

Walsh, B. T. (2017). Assessment of the motivation to use artificial sweetener

among individuals with an eating disorder. Appetite, 109, 131-136.

Shaham, Y., Erb, S., & Stewart, J. (2000). Stress-induced relapse to heroin and cocaine

seeking in rats: A review. Brain Research Reviews, 33(1), 13-33.

50

Shalev, U., Marinelli, M., Baumann, M. H., Piazza, P., & Shaham, Y. (2003). The role of

corticosterone in food deprivation-induced reinstatement of cocaine seeking in

the rat. Psychopharmacology, 168(1-2), 170-176.

Sinha, R. (2008). Chronic stress, drug use, and vulnerability to addiction. Annals of the

New York Academy of Sciences, 1141(1), 105-130.

Sprague, J. E., & Maickel, R. P. (1994). Effects of stress and ebiratide (hoe-427) on

free-choice ethanol consumption: Comparison of lewis and sprague-dawley

rats. Life Sciences, 55(11), 873-878.

Stanhope, K. L., Medici, V., Bremer, A. A., Lee, V., Lam, H. D., Nunez, M. V., . . . Havel,

P. J. (2015). A dose-response study of consuming high-fructose corn syrup-

sweetened beverages on lipid/lipoprotein risk factors for cardiovascular disease

in young adults. American Journal of Clinical Nutrition, 101(6), 1144-1154.

Steinberg, E. E., Boivin, J. R., Saunders, B. T., Witten, I. B., Deisseroth, K., & Janak, P.

H. (2014). Positive reinforcement mediated by midbrain dopamine neurons

requires D1 and D2 receptor activation in the nucleus accumbens. PLoS

One, 9(4).

Tatzer, E., Schubert, M. T., Timischl, W., & Simbruner, G. (1985). Discrimination of

taste and preference for sweet in premature babies. Early Human

Development, 12(1), 23-30.

Thai, P., Tan, E., Tan, W., Tey, T., Kaur, H., & Say, Y. (2011). Sweetness intensity

perception and pleasantness ratings of sucrose, aspartame solutions and cola

among multi-ethnic malaysian subjects. Food Quality and Preference, 22(3), 281-

289.

51

Thomas, S. E., Bacon, A. K., Randall, P. K., Brady, K. T., & See, R. E. (2011). An acute

psychosocial stressor increases drinking in non-treatment-seeking

alcoholics. Psychopharmacology, 218(1), 19-28.

Tolliver, G. A., Sadeghi, K. G., & Samson, H. H. (1988). Ethanol preference following

the sucrose-fading initiation procedure. Alcohol, 5(1), 9-13.

Vengeliene, V., Siegmund, S., Singer, M. V., Sinclair, J. D., Li, T., & Spanagel, R.

(2003). A comparative study on alcohol-preferring rat lines: Effects of deprivation

and stress phases on voluntary alcohol intake. Alcoholism, Clinical and

Experimental Research, 27(7), 1048-1054.

Ventura, A. K., & Mennella, J. A. (2011). Innate and learned preferences for sweet taste

during childhood. Current Opinion in Clinical Nutrition and Metabolic Care, 14(4),

379-384.

Wheeler, R. A., Aragona, B. J., Fuhrmann, K. A., Jones, J. L., Day, J. J., Cacciapaglia,

F., . . . Carelli, R. M. (2011). Cocaine cues drive opposing context-dependent

shifts in reward processing and emotional state. Biological Psychiatry, 69(11),

1067-1074.

White, N. M., & Carr, G. D. (1985). The conditioned place preference is affected by two

independent reinforcement processes. Pharmacology, Biochemistry, and

Behavior, 23(1), 37-42.

World Health Organization. (2009). Global health risks: mortality and burden of disease

attributable to selected major risks. Geneva, World Health Organization.

World Health Organization. (2014). Global status report on alcohol and health. Geneva,

World Health Organization.

52

Wyvell, C. L., & Berridge, K. C. (2000). Intra-accumbens amphetamine increases the

conditioned incentive salience of sucrose reward: Enhancement of reward

"wanting" without enhanced "liking" or response reinforcement. Journal of

Neuroscience, 20(21), 8122-8130.

Yasoshima, Y., & Shimura, T. (2015). A mouse model for binge-like sucrose

overconsumption: Contribution of enhanced motivation for sweetener

consumption. Physiology & Behavior, 138, 154-164.

53

Figure Legends

Figure 1 A. Timeline of procedural events that occurred during Experiment 1. Rats

could self-administer 5% EtOH under multiple schedules of reinforcement, then 25%

HFCS was added to the solution. Following this, a dose-response curve was created by

varying the concentration of EtOH within HFCS, then rats reverted to self-administering

5% EtOH. B. Mean (SEM) infusions earned across 25 days of IO SA of 5% EtOH

(sessions 1 -24) and 5% EtOH + 25% HFCS (session 25). The labels below the x-axis

refer to the schedule of reinforcement. The *** indicates a significance increase from all

IO SA sessions of 5% EtOH, p <0.001. C. Mean (SEM) infusions earned across

sessions 26 to 45 during which the concentration of EtOH in the 25% HFCS solution

was manipulated to create a dose-response (intake) curve: 5%, 10%, 20%, 5%. Animals

were tested on a FR3 schedule of reinforcement. The *** indicates a significance

decrease from sessions 33 (5%), 37 (10%) and 45 (5%), p <0.001. D. Mean (SEM)

infusions rats earned on the last IO SA acquisition day of 5% EtOH prior to the addition

of HFCS (session 20), compared to the four sessions following HFCS removal

(sessions 46 – 49). The * indicates a significant increase from Pre-HFCS (session 20), p

<0.05. The ** indicates a significant increase from Pre-HFCS, p< 0.01.

Figure 2 A. Timeline of procedural events that occurred during Experiment 2. Rats

could self-administer 5% EtOH in 0.1% SACC. Following this, a dose-response curve

was created by varying the concentration of EtOH within SACC. On the final session,

rats could self-administer 25% HFCS. B. Mean (SEM) infusions earned across 17

sessions of IO SA of 5% EtOH in 0.1% SACC. The labels below the x-axis refer to the

schedule of reinforcement. C. Mean (SEM) infusions earned during acquisition for 5%

54

EtOH (data taken from Experiment 1; session 20) compared to 5% EtOH + 0.1% SACC

(data taken from Experiment 2; session 17). D. Mean (SEM) infusions earned across

sessions 14 - 29, during which the concentration of EtOH in 0.1% SACC solution was

manipulated to create a dose-response (intake) curve: 5%, 10%, 20%, 5%. The ***

indicates a significance difference from session 17 (5% EtOH). E. Mean (SEM)

infusions rats earned on the last day each concentration (5%, 10%, 20%, 5%) of EtOH

was presented in the SACC solution and in the HFCS solution (Experiment 1) during the

dose-response (intake) curve. The *** indicates a significance difference from Exp 1 to

Exp 2 solutions.

Figure 3 A. Timeline of procedural events that occurred during Experiment 3. Rats

could self-administer 10% EtOH in HFCS during acquisition, then this behaviour was

extinguished by replacing sweetened EtOH reinforcement with reverse osmosis water

deliveries. Following extinction, a cue-reinstatement test was used to test drug-seeking

for the drug-paired cue light. Following this, two resumption tests were used to test the

resumption of 10% EtOH and 25% HFCS (across two counterbalanced sessions) under

conditions of food restriction and food deprivation. B. Mean (SEM) infusions earned

across 17 days of IO SA of 10% EtOH in 25% HFCS. The labels below the x-axis refer

to the schedule of reinforcement. The * indicates a significant difference from session 1.

C. Mean (SEM) active lever responses made on the last day of IO SA acquisition

(Baseline; B) and the following five extinction sessions. The * indicates a significant

decrease from Baseline, p < 0.001. The # indicates a significant decrease from Ext1, p

<0.001. D. Mean (SEM) active lever responses made on the tests of cue reinstatement

between food restricted and food deprived rats. The * indicates a significant increase in

55

food restricted relative to food deprived rats. E. Mean (SEM) infusions earned during the

tests of EtOH and HFCS resumption across food restricted and food deprived rats. The

** indicates a significant difference from extinction responding within stressed rats, p <

0.01. The # indicates a significant difference from cue-reinstatement responding within

non-stressed animals.

Figure 4 A. Timeline of procedural events that occurred during Experiment 4. Each

taste reactivity (TR) session tested EtOH and HFCS palatability separately over a two-

min liquid exposure. Please see Figure 3 A for a more detailed explaoination of the IO

SA protocol used. B. Mean (SEM) infusions earned during the test of 10% EtOH + 25%

HFCS resumption across food restricted and food deprived rats. The * indicates a

significant difference from food restriction conditions, p < 0.05. C. Mean (SEM) hedonic

taste responses elicited to EtOH and HFCS during baseline (pre-acquisition) and post-

acquisition taste reactivity testing. The * indicates a significant increase from baseline

responding, p < 0.05. The *** indicates a significant increase from baseline responding,

p < 0.001. D. Mean (SEM) hedonic taste responses elicited to EtOH and HFCS across

food restricted and food deprived rats. The *** indicates a significant increase from food

restriction conditions, p < 0.001.

56

Figure 1.

57

Figure 2.

58

Figure 3.

59

Figure 4.

60

General Discussion

Summary of results

Excessive alcohol consumption by young, naive drinkers is prevelant and has

been associated with an increased risk for developing alcohol use disorders later in life

(Chassin, Pitts, & Prost, 2002; Albers et al., 2015). Furthermore, the addition of

sweeteners to alcoholic beverages have been implicated in increasing alcohol intake of

naïve drinkers (Albers et al., 2015). Thus, the current study sought to investigate

whether the addition of HFCS to an EtOH solution could alter EtOH’s intake, hedonic

taste, and sensitivity to food deprivation stress in naïve rats. HFCS caused a drastic

increase in EtOH intake across multiple concentrations; an effect that was not observed

by the addition of a non-caloric sweetener (SACC). HFCS also altered rat’s sensitivity to

the effect of food deprivation stress on EtOH consumption. More specifically, food

deprivation stress increased EtOH intake only when HFCS was mixed in the solution. In

terms of EtOH and HFCS palatability, our study revealed two important findings. First,

multiple exposures to 10% EtOH in 25% HFCS increased the palatability of 10% EtOH

and 25% HFCS. Second, food deprivation stress increased the palatability of HFCS, but

did not alter the palatability of EtOH. Taken together with the results from self-

administration, we suggest that the increased palatability of HFCS under food

deprivation stress may contribute to the increased motivation to seek HFCS under

stressful operant conditions.

Methodological limitations

One methodological limitation to consider when interpreting the results of the

presented study is the selective use of male rats throughout all experiments. Male rats

61

were chosen because our laboratory has already established the consumption patterns

of HFCS in the home-cage and during operant IO SA. This limits the generalizability of

our findings since differences in alcohol intake and alcohol preferences have been

reported across male and female rodents. Generally, females tend to consume larger

quantities, and demonstrate a higher preference for alcohol in comparison to males

(Lancaster & Spiegel, 1992; Lancaster, Brown, Coker, Elliot, & Wren, 1996). In

corroboration with these behavioural findings, female rats show increased

responsiveness to mesolimbic dopamine stimulation by EtOH exposure (Blanchard,

Steindorf, Wang, & Glick, 1993). Human research has also indicated that women reach

a higher peak blood alcohol concentration (BAC) than men after consuming equivalent

amounts of EtOH (Sutker, Tabakoff, Goist, & Randall, 1983). These findings have been

suggested to reflect the differences in EtOH pharmacokinetics between the two sexes

(Taylor, Dolhert, Friedman, Mumenthaler, Yesavage, 1996; Mumenthaler, Taylor,

O’Hara, Yesavage, 1999).

In addition to differences in alcohol intake and preference, the effect of stress on

EtOH intake might also differ between female and male rodents. For example, restraint

stress has been shown to increase homecage EtOH consumption in male mice, while

decreasing consumption in females (Chester, Gustavo, Demaria, & Finegan, 2006).

Taken together, these results highlight the importance of using both males and females

while investigating rodent models of EtOH intake.

Although our laboratory has frequently studied the reinforcing properties of

HFCS, the current experiments represent our first attempt to assess ethanol

reinforcement. As such, our laboratory was not adequately prepared to measure the

62

blood alcohol concentration (BAC) of rats throughout IO SA. During operant SA of

EtOH in rats, BACs are frequently assessed in addition to the total amount of EtOH

consumed (Doherty & Gonzales, 2015; Carnicella, Yowell, & Ron, 2011). Using BACs

as a biomarker of EtOH consumption is necessary to provide a measure of EtOH

exposure that accounts for other factors that have been shown to affect EtOH

consumption in animals (i.e. patterns of drinking, metabolism, sex differences). Thus,

our experiments could have drawn stronger conclusions about the effects of HFCS on

EtOH consumption if BACs were presented along with consumption data. However,

when the consumption patterns of animals responding for 10% EtOH in 25% HFCS

were calculated in ten-minute time bins (Experiment 1), rats consumed 0.91 g/kg within

the first 30 minutes of each session. Briones and Woods (2013) found that home-cage

self-administration of approximately 0.75 g/kg EtOH in rats produced BACs greater than

0.08 g%, which constitutes as a binge consumption pattern according to the National

institute of Alcohol Abuse and Alcoholism (2015). Therefore, although the current study

lacked measures of BACs throughout the IO SA of EtOH + HFCS, we are confident that

animals consumed quantities of EtOH that produced recognized levels of binge

intoxication.

Future directions

This study could be expanded upon by future research. As previously mentioned,

one limitation of the presented experiments is the inability to draw conclusions about

how HFCS changes the relative reinforcing value of EtOH. A particularly useful way to

measure the reinforcing value of a stimulus while using an operant self-administration

model is by applying a progressive-ratio schedule of reinforcement. When a

63

progressive-ratio schedule is applied, the amount of effort required to earn

reinforcement successively increases across each trial, until responding for the stimulus

ceases and a ‘breakpoint’ has been achieved (Jones & Comer, 2013; Panlilio &

Goldberg, 2007). Given that breakpoints quantify an organism’s persistence/desire to

achieve reinforcement, with higher breakpoints associated with higher persistence, they

can be used to indicate the relative reinforcing value of different stimuli. Furthermore,

the application of a progressive ratio schedule also stabilizes possible confounds

between solutions used during operant self-administration such as, levels of satiety or

intoxication.

Choice procedures are another method that can be employed to delineate

changes in a stimulus’ reinforcing value; in fact, some researchers argue choice

procedures are the only method by which changes in the reinforcing value of a stimulus

can be determined (Panlilio & Goldberg, 2007). During choice testing, participants are

required to choose between a fixed magnitude of the particular reinforcer of interest, or

a scaling amount of an alternative reinforcer. The point at which the participant stops

choosing the reinforcer of interest over the alternative reinforcer is then used to

compare the relative reinforcing value of the reinforcer of interest (in comparison to the

alternative).

Although the use of progressive ratio or choice testing could allow us to make

stronger inferences about changes in the reinforcing efficacy of EtOH, both procedures

would be complicated to employ as rats do not self-administer reliable amount of EtOH

without the use of sucrose-fading or lengthy intitiation procedures (as reviewed by

Samson, 1986; Samson, Pfeffer, & Tolliver, 1988). Ultimetly the use of sucrose-fading

64

would confound the variable of interest; that is, changes in EtOH reinforcing efficacy as

a result of HFCS pairings. Furthermore, alternate initiation procedures could take

upwards of 6-weeks, and do not lead to strong responding for EtOH alone. Therefore,

although our lab has considered alternate ways to investigate the changes in EtOH’s

reinforcing value, there are multiple limitations in using these methods to measure a

reinforcer that is not reliably self-administered on its own (e.x. EtOH).

Our laboratory is also interested in examining the role of arginine vasopressin in

EtOH intake, palatability and persistence of responding under conditions of food

deprivation stress. Arginine vasopressin (AVP) is synthesized in the supraoptic nucleus

of the hypothalamus and acts as a key regulator of the hypothalamic-pituitary-adrenal

(HPA) axis, the body’s central stress response system (Aguilera & Rabadan-Diehl,

2000; Tanoue et al., 2004). More specifically, following exposure to stress, AVP binds to

V1b receptors (V1bR) in the anterior pituitary to promote the secretion of

adrenocorticotropic hormone (ACTH) into the bloodstream. ACTH subsequently travels

down to the adrenal cortex to promote the synthesis and release of glucocorticoids (see

Beurel & Nemeroff, 2014 for a review). Correspondingly, V1bR antagonists decrease

multiple anxiogenic behaviours in animal models (Griebel et al., 2002; Stemmelin,

Lukovic, Salome, & Griebel., 2005).

Given that AVP modulates anxiogenic responding, it may contribute to stress-

induced relapse. Indeed, AVP secretion is heightened during periods of drug withdrawal

in humans and animals. For example, Eisenhofer, Lambie, Whiteside, and Johnson

(1985) measured AVP levels in alcoholic patients during a drug detoxification program.

Patients displaying withdrawal symptoms had significantly elevated urine and plasma

65

AVP levels in comparison to patients without withdrawal symptomology, or healthy

controls. Furthermore, rats show heightened AVP mRNA gene expression during

periods of acute heroin and cocaine withdrawal (Zhou, Leri, Cummins, Hoeschele, &

Kreek, 2008; Zhou et al., 2005).

There are few studies that have investigated the effect of V1bR antagonists on

relapse-like responding in animals. Zhou, Leri, Cummins, Hoeschele, and Kreek (2008)

found that the administration of a V1bR antagonist (1 mg/kg and 30 mg/kg;

SSR149415) decreased stress induced relapse-like responding for heroin in rats.

Interestingly, the function of AVP in modulating relapse-like responding for alcohol is

unknown. It would be interesting to investigate whether the V1bR antagonist

SSR149415 would decrease stress-induced increases in resumption responding for

EtOH + HFCS and HFCS.

Conclusions

In conclusion, results from Experiment 1 and Experiment 2 suggest that the

addition of a sweet and calorically dense sweetener (HFCS) drastically elevated EtOH

intake in rats, in comparison to an equally palatable, yet non-caloric sweetener.

Furthermore, when HFCS was removed from the EtOH solution, rats demonstrated

increased EtOH intake levels then what was observed prior to the HFCS pairings.

Finally, Experiment 3 and Experiment 4 demonstrated that food deprivation stress only

increased EtOH intake when HFCS was mixed into the solution. Furthermore, results

from taste reactivity measures suggest that food deprivation stress causes increase in

HFCS palatability, which may explain the observed self-administration data. Taken

together, our results suggest that HFCS increases EtOH intake under normal and

66

stressful conditions, and should be reconsidered as an acceptable addition to alcoholic

beverages.

67

References Ackroff, K., & Sclafani, A. (2011). Flavor preferences conditioned by post-oral infusion of

monosodium glutamate in rats. Physiology & Behavior, 104(3), 488-494.

Albers, A. B., Siegel, M., Ramirez, R. L., Ross, C., DeJong, W., & Jernigan, D. H.

(2015). Flavored alcoholic beverage use, risky drinking behaviors, and adverse

outcomes among underage drinkers: Results from the ABRAND study. American

Journal of Public Health, 105(4), 810-815.

Agmo, A., & Marroquin, E. (1997). Role of gustatory and postingestive actions of

sweeteners in the generation of positive affect as evaluation by place preference

conditioning. Appetite, 29(3), 269-289.

Aguilera, G., & Rabadan-Diehl, C. (2000). Vasopressinergic regulation of the

hypothalamic-pituitary-adrenal axis: Implications for stress

adaptation. Regulatory Peptides, 96(1-2), 23-29.

Anderson, D. A., Shapiro, J. R., Lundgren, J. D., Spataro, L. E., & Frye, C. A. (2002).

Self-reported dietary restraint is associated with elevated levels of salivary

cortisol. Appetite, 38(1), 13-17.

Archie, S., Zangeneh-Kazemi, A., & Akhtar-Danesh, N. (2012). Concurrent binge

drinking and depression among canadian youth: Prevalence, patterns, and

suicidality. Alcohol, 46(2), 165-72.

Avena, N. M., Long, K. A., & Hoebel, B. G. (2005). Sugar-dependent rats show

enhanced responding for sugar after abstinence: Evidence of a sugar deprivation

effect. Physiology & Behavior, 84(3), 359-362.

68

Barlow, P., McKee, Basu, & Stuckler, (2017). Impact of the north american free trade

agreement on high-fructose corn syrup supply in canada: A natural experiment

using synthetic control methods. Canadian Medical Association.Journal, 189(26),

E881-E887.

Beurel, E., & Nemeroff, C. B. (2014). Interaction of stress, corticotropin-releasing factor,

arginine vasopressin and behaviour. Current Topics in Behavioral

Neurosciences, 18, 67-80.

Bienkowski, P., Koros, E., Kostowski, W., & Bogucka-Bonikowska, A. (2000).

Reinstatement of ethanol seeking in rats: Behavioral analysis. Pharmacology

Biochemistry and Behavior, 66(1), 123-128.

Black, J., Belluzzi, J. D., & Stein, L. (1985). Reinforcement delay of one second

severely impairs acquisition of brain self-stimulation. Brain Research, 359(1-2),

113-119.

Blanchard, B. A., Steindorf, S., Wang, S., & Glick, S. D. (1993). Sex differences in

ethanol-induced dopamine release in nucleus accumbens and in ethanol

consumption in rats. Alcoholism, Clinical and Experimental Research, 17(5), 968-

973.

Bouton, M. E., Winterbauer, N. E., & Todd, T. P. (2012). Relapse processes after the

extinction of instrumental learning: Renewal, resurgence, and reacquisition.

Behavioural Processes, 90(1), 130-141.

Bray, G. A., Nielsen, S. J., & Popkin, B. M. (2004). Consumption of high-fructose corn

syrup in beverages may play a role in the epidemic of obesity. The American

Journal of Clinical Nutrition, 79(4), 537-543.

69

Bray, G. A. (2013). Energy and fructose from beverages sweetened with sugar or high-

fructose corn syrup pose a health risk for some people. Advances in

Nutrition, 4(2), 220-225.

Briones, T. L., & Woods, J. (2013). Chronic binge-like alcohol consumption in

adolescence causes depression-like symptoms possibly mediated by the effects

of BDNF on neurogenesis. Neuroscience, 254, 324-334.

Brown, S. A., Vik, P. W., McQuaid, J. R., Patterson, T. L., Irwin, M. R., & Grant, I.

(1990). Severity of psychosocial stress and outcome of alcoholism

treatment. Journal of Abnormal Psychology, 99(4), 344-348.

Brown, S. A., Vik, P. W., Patterson, T. L., Grant, I., & Schuckit, M. A. (1995). Stress,

vulnerability and adult alcohol relapse. Journal of Studies on Alcohol, 56(5), 538-

545.

Bulik, C. M., & Brinded, E. C. (1993). The effect of food deprivation on alcohol

consumption in bulimic and control women. Addiction, 88(11), 1545-1551.

Carnicella, S., Yowell, Q. V., & Ron, D. (2011). Regulation of operant oral ethanol self-

administration: A dose-response curve study in rats. Alcoholism, Clinical and

Experimental Research, 35(1), 116-125.

Carr, K. D. (2007). Chronic food restriction: Enhancing effects on drug reward and

striatal cell signaling. Physiology & Behavior, 91(5), 459-472.

Carroll, M. E. (1985). The role of food deprivation in the maintenance and reinstatement

of cocaine-seeking behavior in rats. Drug and Alcohol Dependence, 16(2), 95-

109.

70

Chassin, L., Pitts, S. C., & Prost, J. (2002). Binge drinking trajectories from adolescence

to emerging adulthood in a high-risk sample: Predictors and substance abuse

outcomes. Journal of Consulting and Clinical Psychology, 70(1), 67-78.

Chaudhri, N., Sahuque, L. L., Cone, J. J., & Janak, P. H. (2008). Reinstated ethanol-

seeking in rats is modulated by environmental context and requires the nucleus

accumbens core. European Journal of Neuroscience, 28(11), 2288-2298.

Chester, J. A., Gustavo de, P. B., DeMaria, A., & Finegan, A. (2006). Different effects of

stress on alcohol drinking behaviour in male and female mice selectively bred for

high alcohol preference. Alcohol and Alcoholism: International Journal of the

Medical Council on Alcoholism, 41(1), 44.

Cooney, N. L., Litt, M. D., Morse, P. A., Bauer, L. O., & Gaupp, L. (1997). Alcohol cue

reactivity, negative-mood reactivity, and relapse in treated alcoholic men. Journal

of Abnormal Psychology, 106(2), 243-250.

Copeland, J., Stevenson, R. J., Gates, P., & Dillon, P. (2007). Young australians and

alcohol: The acceptabllity of ready-to-drink (RTD) alcoholic beverages among 12-

30-year-olds. Addiction, 102(11), 1740-1746.

Crombag, H. S., Bossert, J. M., Koya, E., & Shaham, Y. (2008). Context-induced

relapse to drug seeking: a review. Philosophical Transactions of the Royal

Society of London. Series B, Biological Sciences, 363(1507), 3233-3243.

Daniels, S., Marshall, P., & Leri, F. (2016). Alterations of naltrexone-induced

conditioned place avoidance by pre-exposure to high fructose corn syrup or

heroin in sprague-dawley rats. Psychopharmacology, 233(3), 425-433.

71

D'Cunha, T.,M., Sedki, F., Macri, J., Casola, C., & Shalev, U. (2013). The effects of

chronic food restriction on cue-induced heroin seeking in abstinent male rats.

Psychopharmacology, 225(1), 241-250.

DeWit, D. J., Adlaf, E. M., Offord, D. R., & Ogborne, A. C. (2000). Age at first alcohol

use: A risk factor for the development of alcohol disorders. The American Journal

of Psychiatry, 157(5), 745-750.

Dietze, S., Lees, K. R., Fink, H., Brosda, J., & Voigt, J. (2016). Food deprivation, body

weight loss and anxiety-related behavior in rats. Animals, 6(1), 4.

Doherty, J. M., & Gonzales, R. A. (2015). Operant self-administration of sweetened

ethanol and time course of blood ethanol levels in adolescent and adult male

long-evans rats. Alcoholism, Clinical and Experimental Research, 39(3), 485-

495.

Domingos, A. I., Vaynshteyn, J., Voss, H. U., Ren, X., Gradinaru, V., Zang, F., . . .

Friedman, J. (2011). Leptin regulates the reward value of nutrient. Nature

Neuroscience, 14(12), 1562-8.

Eisenhofer, G., Lambie, D. G., Whiteside, E. A., & Johnson, R. H. (1985). Vasopressin

concentrations during alcohol withdrawal. British Journal of Addiction, 80(2), 195-

199.

Fidler, T. L., Clews, T. W., & Cunningham, C. L. (2006). Reestablishing an intragastric

ethanol self-infusion model in rats. Alcoholism, Clinical and Experimental

Research, 30(3), 414-428.

72

Filbey, F. M., Claus, E., Audette, A. R., Niculescu, M., Banich, M. T., Tanabe, J., . . .

Hutchison, K. E. (2008). Exposure to the taste of alcohol elicits activation of the

mesocorticolimbic neurocircuitry. Neuropsychopharmacology, 33(6), 1391-1401.

Fox, H. C., Bergquist, K. L., Hong, K., & Sinha, R. (2007). Stress-induced and alcohol

cue-induced craving in recently abstinent alcohol-dependent

individuals. Alcoholism, Clinical and Experimental Research, 31(3), 395-403.

Frank, G. K. W., Oberndorfer, T. A., Simmons, A. N., Paulus, M. P., Fudge, J. L., Yang,

T. T., & Kaye, W. H. (2008). Sucrose activates human taste pathways differently

from artificial sweetener. NeuroImage, 39(4), 1559-1569.

Garland, E. L., Franken, I. H. A., & Howard, M. O. (2012). Cue-elicited heart rate

variability and attentional bias predict alcohol relapse following treatment.

Psychopharmacology, 222(1), 17-26.

Gass, J. T., & Olive, M. F. (2007). Reinstatement of ethanol-seeking behavior following

intravenous self-administration in wistar rats. Alcoholism, Clinical and

Experimental Research, 31(9), 1441-1445.

Griebel, G., Simiand, J., Serradeil-Le Gal, C., Wagnon, J., Pascal, M., Scatton, B., . . .

Soubrie, P. (2002). Anxiolytic- and antidepressant-like effects of the non-peptide

vasopressin V sub(1b) receptor antagonist, SSR149415, suggest an innovative

approach for the treatment of stress-related disorders. Proceedings of the

National Academy of Sciences, 99(9), 6370-6375.

Gruber, E., DiClemente, R. J., Anderson, M. M., & Lodico, M. (1996). Early drinking

onset and its association with alcohol use and problem behavior in late

adolescence. Preventive Medicine, 25(3), 293-300.

73

Guttman, N. (1954). Equal-reinforcement values for sucrose and glucose solutions

compared with equal-sweetness values. Journal of Comparative and

Physiological Psychology, 47(5), 358-361.

Hajnal, A., Smith, G. P., & Norgren, R. (2004). Oral sucrose stimulation increases

accumbens dopamine in the rat. American Journal of Physiology.Regulatory,

Integrative and Comparative Physiology, 286(1), R31-R37.

Hawkins, J. D., Graham, J. W., Maguin, E., Abbott, R., Hill, K. G., & Catalano, R. F.

(1997). Exploring the effects of age of alcohol use initiation and psychosocial risk

factors on subsequent alcohol misuse. Journal of Studies on Alcohol, 58(3), 280-

290.

Higley, A. E., Crane, N. A., Spadoni, A. D., Quello, S. B., Goodell, V., & Mason, B. J.

(2011). Craving in response to stress induction in a human laboratory paradigm

predicts treatment outcome in alcohol-dependent

individuals. Psychopharmacology, 218(1), 121-129.

Highfield, D. A., Mead, A. N., Grimm, J. W., Rocha, B. A., & Shaham, Y. (2002).

Reinstatement of cocaine seeking in 129X1/SvJ mice: Effects of cocaine priming,

cocaine cues and food deprivation. Psychopharmacology, 161(4), 417-424.

Hingson, R. W., Heeren, T., & Winter, M. R. (2006). Age of alcohol-dependence onset:

Associations with severity of dependence and seeking treatment.

Pediatrics, 118(3), e755-e763.

Hogenkamp, P. S., Shechter, A., St-Onge, M., Sclafani, A., & Kissileff, H. R. (2017). A

sipometer for measuring motivation to consume and reward value of foods and

74

beverages in humans: Description and proof of principle. Physiology &

Behavior, 171, 216-227.

Horvitz, J. C. (2000). Mesolimbocortical and nigrostriatal dopamine responses to salient

non-reward events. Neuroscience, 96(4), 651-656.

Jin, H., Rourke, S. B., Patterson, T. L., Taylor, M. J., & Grant, I. (1998). Predictors of

relapse in long-term abstinent alcoholics. Journal of Studies on Alcohol, 59(6),

640-646.

Katner, S. N., Magalong, J. G., & Weiss, F. (1999). Reinstatement of alcohol-seeking

behavior by drug-associated discriminative stimuli after prolonged extinction in

the rat. Neuropsychopharmacology, 20(5), 471-479.

Knight, C. P., Hauser, S. R., Deehan, G. A., Toalston, J. E., McBride, W. J., & Rodd, Z.

A. (2016). Oral conditioned cues can enhance or inhibit ethanol (EtOH)-seeking

and EtOH-relapse drinking by alcohol-preferring (P) rats. Alcoholism, Clinical and

Experimental Research, 40(4), 906-915.

Lancaster, F. E., Brown, T. D., Coker, K. L., Elliott, J. A., & Wren, S. B. (1996). Sex

differences in alcohol preference and drinking patterns emerge during the early

postpubertal period. Alcoholism, Clinical and Experimental Research, 20(6),

1043-1049.

Lancaster, F. E., & Spiegel, K. S. (1992). Sex differences in pattern of drinking.Alcohol

(Fayetteville, N.Y.), 9(5), 415-420.

Lanier, S. A., Hayes, J. E., & Duffy, V. B. (2005). Sweet and bitter tastes of alcoholic

beverages mediate alcohol intake in of-age undergraduates. Physiology &

Behavior, 83(5), 821-831.

75

Le, A. D., Harding, S., Juzytsch, W., Funk, D., & Shaham, Y. (2005). Role of alpha-2

adrenoceptors in stress-induced reinstatement of alcohol seeking and alcohol

self-administration in rats. Psychopharmacology, 179(2), 366-373.

Leeman, R. F., O'Malley, S.,S., White, M. A., & McKee, S. A. (2010). Nicotine and food

deprivation decrease the ability to resist smoking. Psychopharmacology, 212(1),

25-32.

Lenoir, M., Serre, F., Cantin, L., & Ahmed, S. H. (2007). Intense sweetness surpasses

cocaine reward. PloS One, 2(8), 1.

Levy, A., Limebeer, C. L., Ferdinand, J., Shillingford, U., Parker, L. A., & Leri, F. (2014).

A novel procedure for evaluating the reinforcing properties of tastants in

laboratory rats: Operant intraoral self-administration. Journal of Visualized

Experiments, 84, 1.

Levy, A., Marshall, P., Zhou, Y., Kreek, M. J., Kent, K., Daniels, S., . . . Leri, F. (2015).

Fructose: Glucose ratios-A study of sugar self-administration and associated

neural and physiological responses in the rat. Nutrients, 7(5), 3869-3890.

Levy, A., Daniels, S., Hudson, R., Horman, T., Flynn, A., Zhou, Y., & Leri, F. (2018).

Bupropion and naltrexone combination alters high fructose corn syrup self-

administration and gene expression in rats. Neuropharmacology, 135, 547-554.

Liu, X., & Weiss, F. (2002). Additive effect of stress and drug cues on reinstatement of

ethanol seeking: Exacerbation by history of dependence and role of concurrent

activation of corticotropin-releasing factor and opioid mechanisms. The Journal of

Neuroscience, 22(18), 7856-7861.

76

Maccioni, P., Orru, A., Korkosz, A., Gessa, G. L., Carai, M. A. M., Colombo, G., &

Bienkowski, P. (2007). Cue-induced reinstatement of ethanol seeking in

sardinian alcohol-preferring rats. Alcohol, 41(1), 31-39.

Madsen, H. B., & Ahmed, S. H. (2015). Drug versus sweet reward: Greater attraction to

and preference for sweet versus drug cues. Addiction Biology, 20(3), 433-444.

Martin, R., & Weiss, F. (2013). Modeling relapse in animals. Current Topics in

Behavioral Neurosciences, 13, 403-432.

Meyers, A. M., Mourra, D., & Beeler, J. A. (2017). High fructose corn syrup induces

metabolic dysregulation and altered dopamine signaling in the absence of

obesity. PLoS One, 12(12).

McLean, J. A., Barr, S. I., & Prior, J. C. (2001). Cognitive dietary restraint is associated

with higher urinary cortisol excretion in healthy premenopausal women. The

American Journal of Clinical Nutrition, 73(1), 7-12.

Moos, R. H., & Moos, B. S. (2006). Rates and predictors of relapse after natural and

treated remission from alcohol use disorders. Addiction, 101(2), 212-222.

Mosher, J. F., & Johnsson, D. (2005). Flavored alcoholic beverages: An international

marketing campaign that targets youth. Journal of Public Health Policy, 26(3),

326-342.

Mumenthaler, M. S., Taylor, J. L., O'Hara, R., Fisch, H. U., & Yesavage, J. A. (1999).

Effects of menstrual cycle and female sex steroids on ethanol

pharmacokinetics.Alcoholism, Clinical and Experimental Research, 23(2), 250-

255.

77

Nowland, M. H., Hugunin, K. M. S., & Rogers, K. L. (2011). Effects of short-term fasting

in male sprague-dawley rats. Comparative Medicine, 61(2), 138-144.

Oberlin, B. G., Dzemidzic, M., Tran, S. M., Soeurt, C. M., Albrecht, D. S., Yoder, K. K.,

& Kareken, D. A. (2013). Beer flavor provokes striatal dopamine release in male

drinkers: Mediation by family history of

alcoholism. Neuropsychopharmacology, 38(9), 1617-24.

O'Connor, E.,C., Chapman, K., Butler, P., & Mead, A. N. (2011). The predictive validity

of the rat self-administration model for abuse liability. Neuroscience &

Biobehavioral Reviews, 35(3), 912-938.

Panlilio, L. V., & Goldberg, S. R. (2007). Self-administration of drugs in animals and

humans as a model and an investigative tool. Addiction, 102(12), 1863-1870.

Perry, C. J., Zbukvic, I., Kim, J. H., & Lawrence, A. J. (2014). Role of cues and contexts

on drug-seeking behaviour. British Journal of Pharmacology, 171(20), 4636-

4672.

Petrakis, I. L., Gonzalez, G., Rosenheck, R., & Krystal, J. H. (2002). Comorbidity of

alcoholism and psychiatric disorders: An overview. Alcohol Research and

Health, 26(2), 81-89.

Preston, K. L., & Epstein, D. H. (2011). Stress in the daily lives of cocaine and heroin

users: Relationship to mood, craving, relapse triggers, and cocaine use.

Psychopharmacology, 218(1), 29-37.

Rada, P., Avena, N. M., & Hoebel, B. G. (2005). Daily bingeing on sugar repeatedly

releases dopamine in the accumbens shell. Neuroscience, 134(3), 737-744.

78

Regier, D. A., Farmer, M. E., Rae, D. S., Locke, B. Z., Keith, S. J., Judd, L. L., &

Goodwin, F. K. (1990). Comorbidity of mental disorders with alcohol and other

drug abuse: results from the epidemiologic catchment area (ECA) study. Journal

of the American Medical Association, 264(19), 2511-2518.

Rehm, J., Mathers, C., Popova, S., Thavorncharoensap, M., Teerawattananon, Y., &

Patra, J. (2009). Alcohol and global health 1: Global burden of disease and injury

and economic cost attributable to alcohol use and alcohol-use disorders. The

Lancet, 373(9682), 2223-2233.

Robbins, T. W., & Everitt, B. J. (1996). Neurobehavioural mechanisms of reward and

motivation. Current Opinion in Neurobiology, 6(2), 228-236.

Romanus, G. (2000). Alcopops in sweden--a supply side initiative. Addiction, 95 Suppl

4, S609-S619.

Samson, H. H. (1986). Initiation of ethanol reinforcement using a sucrose-substitution

procedure in food- and water-sated rats. Alcoholism, Clinical and Experimental

Research, 10(4), 436-442.

Samson, H. H., Pfeffer, A. O., & Tolliver, G. A. (1988). Oral ethanol self-administration

in rats: Models of alcohol-seeking behavior. Alcoholism, Clinical and

Experimental Research, 12(5), 591-598.

Schebendach, J., Klein, D. A., Mayer, L. E. S., Attia, E., Devlin, M. J., Foltin, R. W., &

Walsh, B. T. (2017). Assessment of the motivation to use artificial sweetener

among individuals with an eating disorder. Appetite, 109, 131-136.

79

Schulteis, G., Hyytiä, P., Heinrichs, S. C., & Koob, G. F. (1996). Effects of chronic

ethanol exposure on oral self-administration of ethanol or saccharin by wistar

rats. Alcoholism, Clinical and Experimental Research, 20(1), 164-171.

Sclafani, A., & Ackroff, K. (2017). Flavor preferences conditioned by nutritive and non-

nutritive sweeteners in mice. Physiology & Behavior, 173, 188-199.

Shaham, Y., Erb, S., & Stewart, J. (2000). Stress-induced relapse to heroin and cocaine

seeking in rats: A review. Brain Research Reviews, 33(1), 13-33.

Shaham, Y., Shalev, U., Lu, L., de Witt, H., Stewart, J. (2002). The reinstatement model

of drug relapse: history, methodology and major findings. Psychopharmacology,

168 (1), 3-20.

Shalev, U., Yap, J., & Shaham, Y. (2001). Leptin attenuates acute food deprivation-

induced relapse to heroin seeking. The Journal of Neuroscience: The Official

Journal of the Society for Neuroscience, 21(4), 1.

Sinha, R. (2008). Chronic stress, drug use, and vulnerability to addiction. Annals of the

New York Academy of Sciences, 1141(1), 105-130.

Sinha, R., Fox, H. C., Hong, K. A., Hansen, J., Tuit, K., & Kreek, M. J. (2011). Effects of

adrenal sensitivity, stress- and cue-induced craving, and anxiety on subsequent

alcohol relapse and treatment outcomes. Archives of General Psychiatry, 68(9),

942-952.

Smith, S. G., & Davis, W. M. (1974). Intravenous alcohol self-administration in the rat.

Pharmacological Research Communications, 6(4), 379-402.

Stanhope, K. L., Medici, V., Bremer, A. A., Lee, V., Lam, H. D., Nunez, M. V., . . . Havel,

P. J. (2015). A dose-response study of consuming high-fructose corn syrup-

80

sweetened beverages on lipid/lipoprotein risk factors for cardiovascular disease

in young adults. American Journal of Clinical Nutrition, 101(6), 1144-1154.

Steinberg, E. E., Boivin, J. R., Saunders, B. T., Witten, I. B., Deisseroth, K., & Janak, P.

H. (2014). Positive reinforcement mediated by midbrain dopamine neurons

requires D1 and D2 receptor activation in the nucleus accumbens. PLoS

One, 9(4).

Stemmelin, J., Lukovic, L., Salome, N., & Griebel, G. (2005). Evidence that the lateral

septum is involved in the antidepressant-like effects of the vasopressin V1b

receptor antagonist, SSR149415. Neuropsychopharmacology, 30(1), 35-42.

Substance Abuse and Mental Health Services Administration. (2015). National Survey

on Drug Use and Health (NSDUH). Table 2.20A: Alcohol Use, Binge, Alcohol

Use, and Heavy Alcohol Use in Past Month, by Detailed Age Category: Numbers

in Thousands. Rockville, MD.

Sutker, P. B., Tabakoff, B., Goist, K. C., & Randall, C. L. (1983). Acute alcohol

intoxication, mood states and alcohol metabolism in women and men.

Pharmacology, Biochemistry, and Behavior, 18 Suppl 1, 349-354.

Tatzer, E., Schubert, M. T., Timischl, W., & Simbruner, G. (1985). Discrimination of

taste and preference for sweet in premature babies. Early Human

Development, 12(1), 23-30.

Tanoue, A., Ito, S., Honda, K., Oshikawa, S., Kitagawa, Y., Koshimizu, T., . . .

Tsujimoto, G. (2004). The vasopressin V1b receptor critically regulates

hypothalamic-pituitary-adrenal axis activity under both stress and resting

conditions. The Journal of Clinical Investigation, 113(2), 302-309.

81

Taylor, J. L., Dolhert, N., Friedman, L., Mumenthaler, M., & Yesavage, J. A. (1996).

Alcohol elimination and simulator performance of male and female aviators: A

preliminary report. Aviation, Space and Environmental Medicine, 67(5), 407-413.

Thai, P., Tan, E., Tan, W., Tey, T., Kaur, H., & Say, Y. (2011). Sweetness intensity

perception and pleasantness ratings of sucrose, aspartame solutions and cola

among multi-ethnic malaysian subjects. Food Quality and Preference, 22(3), 281-

289.

Thorndike, E. L. (1898). Animal intelligence: An experimental study of the associative

processes in animals. Psychological Monographs: General and Applied, 2(4), i-

109.

Tobin, S., Newman, A. H., Quinn, T., & Shalev, U. (2009). A role for dopamine D1-like

receptors in acute food deprivation-induced reinstatement of heroin seeking in

rats. The International Journal of Neuropsychopharmacology, 12(2), 217-26.

Ventura, A. K., & Mennella, J. A. (2011). Innate and learned preferences for sweet taste

during childhood. Current Opinion in Clinical Nutrition and Metabolic Care, 14(4),

379-384.

Waller, M. B., McBride, W. J., Gatto, G. J., Lumeng, L., & Li, T. K. (1984). Intragastric

self-infusion of ethanol by ethanol-preferring and -nonpreferring lines of rats.

Science, 225(4657), 78-80.

Wheeler, R. A., Aragona, B. J., Fuhrmann, K. A., Jones, J. L., Day, J. J., Cacciapaglia,

F., . . . Carelli, R. M. (2011). Cocaine cues drive opposing context-dependent

shifts in reward processing and emotional state. Biological Psychiatry, 69(11),

1067-1074.

82

White, N. M., & Carr, G. D. (1985). The conditioned place preference is affected by two

independent reinforcement processes. Pharmacology, Biochemistry, and

Behavior, 23(1), 37-42.

Wise, R. A., & Rompre, P. P. (1989). Brain dopamine and reward. Annual Review of

Psychology, 40, 191-225.

Willcocks, A. L., & McNally, G. P. (2013). The role of medial prefrontal cortex in

extinction and reinstatement of alcohol-seeking in rats. The European Journal of

Neuroscience, 37(2), 259-268.

Williams, B. A. (1976). The effects of unsignalled delayed reinforcement. Journal of the

Experimental Analysis of Behavior, 26(3), 441-449.

World Health Organization. (2009). Global health risks: mortality and burden of disease

attributable to selected major risks. Geneva, World Health Organization.

World Health Organization. (2014). Global status report on alcohol and health. Geneva,

World Health Organization.

Wyvell, C. L., & Berridge, K. C. (2000). Intra-accumbens amphetamine increases the

conditioned incentive salience of sucrose reward: Enhancement of reward

"wanting" without enhanced "liking" or response reinforcement. Journal of

Neuroscience, 20(21), 8122-8130.

Yasoshima, Y., & Shimura, T. (2015). A mouse model for binge-like sucrose

overconsumption: Contribution of enhanced motivation for sweetener

consumption. Physiology & Behavior, 138, 154-164.

83

Yu, G., Chen, H., & Sharp, B. M. (2014). Amplified reacquisition of nicotine self-

administration in rats by repeated stress during abstinence.

Psychopharmacology, 231(16), 3189-3195.

Zhou, Y., Leri, F., Cummins, E., Hoeschele, M., & Kreek, M. J. (2008). Involvement of

arginine vasopressin and V1b receptor in heroin withdrawal and heroin seeking

precipitated by stress and by heroin. Neuropsychopharmacology, 33(2), 226-236.

Zhou, Y., Bendor, J. T., Yuferov, V., Schlussman, S. D., Ho, A., & Kreek, M. J. (2005).

Amygdalar vasopressin mRNA increases in acute cocaine withdrawal: Evidence

for opioid receptor modulation. Neuroscience, 134(4), 1391-1397.