Running head: SEX AND INCUBATION OF CRAVING 1

The Effects of Sex on the Incubation of Craving

Meagan E. Malesic

Bloomsburg University of Pennsylvania

SEX AND INCUBATION OF CRAVING 2

Abstract

It has been widely accepted by researchers that explicit cues within an environment largely

influence the phenomenon of the incubation of craving. However, the effects of sex differences

on the amount of craving across the incubation period following withdrawal has not yet been

thoroughly researched. Therefore, 6 male and 6 female adult Sprague-Dawley rats were

conditioned to press a lever in order to receive a sucrose pellet across a 2-week self-

administration period. Concurrently, whenever the lever was pressed, visual and auditory cues

were also triggered. Following the self-administration period, the rats experienced 3 extinction

tests on Day 1, Day 18, and Day 22 to measure their amount of craving. Results revealed that

there was a significant effect of sex on the amount of craving, with female subjects portraying a

significantly higher amount of craving than the male subjects. Although further research is still

required to determine if sex differences influence the incubation of craving phenomenon, the

current study suggests that sex does indeed significantly affect amount of craving.
SEX AND INCUBATION OF CRAVING 3

The Effects of Sex on the Incubation of Craving

Jeffrey Grimm reported the first paper on the incubation of craving after observing that

following the initial withdrawal date from an addictive substance, the subjects amount of

craving tended to increase progressively across the weeks following, rather than decrease (Lu,

Grimm, Dempsey, & Shaham, 2004; Grimm, Hope, Wise, & Shaham, 2001). Following this

discovery, additional researchers began to study this phenomenon, and realized that external

stimuli which subjects tend to associate with the addictive substance act as cues, leading to an

increase in craving (Cooper, Barnea-Ygael, Levy, Shaham, & Zangen, 2007; Madesen, Zbukvic,

Luikinga, Lawrence, & Kim, 2016; Li et al., 2014). This increase in craving due to specific cues

in the environment often results in high levels of relapse for the individuals addicted to the

substance.

Additionally, research conducted on male and female rats has suggested that sex

differences may be present in multiple facets of addiction. Recent evidence implies that females

may become dependent upon an addictive substance quicker than males. However, while

females may also be more prone to relapsing upon encountering environmental cues than males,

males are overall more susceptible to addiction (Sanchez, Moore, Brunzell, & Lynch, 2013;

Wetherill, Jagannathan, Hager, Childress, & Franklin, 2015; Klump, Racine, Hildebrant, & Sisk,

2015). Therefore, it is evident that sex differences between males and females cause different

reactions to addictive substances and cues (Kosten & Zhang, 2008; Reichel, Chan, Ghee, & See,

2012). Recognizing these sex differences may play a large role in the formation of successful

treatment strategies for addiction recovery for both males and females.

Grimm, Hope, Wise, and Shaham (2001) researched the phenomenon of the incubation of

craving in regards to cocaine withdrawal. The study trained rats to associate the cues of lever
SEX AND INCUBATION OF CRAVING 4

pressing and an auditory tone with the deliverance of cocaine, and the cues remained present in

the environment even once the cocaine deliverance ceased. The results of the study indicated

that levels of craving remained significantly heightened in rats for at least two months following

the primary withdrawal period, suggesting that explicit cues encourage the phenomenon of

heightened craving. Additionally, Madsen, Zbukvic, Luikinga, Lawrence, and Kim (2016)

investigated whether cue-extinction therapy during the abstinence period would reduce the

incubation of craving; interestingly, the results study did indeed suggest that rats exposed to cue-

extinction therapy showed significantly fewer signs of craving than rats who were left in the

original environment, exposed to the cocaine reinforcement cues.

In regards to sex differences within addiction, Reichel, Chan, Ghee, and See (2012)

researched female responses to methamphetamine (meth) addiction in comparison to male

responses. The results of the study suggested that female rats increased their intake of meth

quicker than the males, as well as suffered from a greater relapse to the meth-seeking tendencies.

Additionally, Sanchez, Moore, Brunzell, and Lynch (2013) aimed to discover whether the same

exercise routine which decreased nicotine craving in male rats would be beneficial in the

reduction of craving for female rats. Interestingly, the treatment, while successful for the males,

showed no significant reduction of craving for females.

It is apparent that the above studies (Grimm et al., 2001; Madsen et al., 2016; Reichel et

al., 2012; Sanchez et al., 2013) suggest not only that the phenomenon of the incubation of

craving is largely attributed to explicit cues associated with the addictive substance within the

environment, but also that sex differences largely influence different reactions and tendencies of

addiction. However, there is limited evidence to suggest if sex differences have any specific

effect on the incubation of craving phenomenon. Therefore, in the present study we tested the
SEX AND INCUBATION OF CRAVING 5

effects of sex on the amount of craving following the withdrawal period across three separate

time points. Six male and six female adult Sprague-Dawley rats were used in the study. Each rat

was conditioned to press a lever in order to receive a sucrose pellet. Whenever the lever was

pressed, an auditory tone was activated and a series of LED lights would flash within the

chamber. Following a two-week period of self-administration, the rats entered into the

withdrawal period, during which the lever no longer delivered sugar pellets when pressed;

however, whenever the lever was activated, the cues of the tone and the lights would still occur.

Three extinction tests were run, during which craving was measured by the number of times the

rats activated the lever as a means of food seeking. The extinction tests were run one day, 18

days, and 22 days following the final day of self-administration. Because of the results of

Reichel et al. (2012), which suggested that female rats were more susceptible to both a higher

rate of addiction and relapse than male rats, it was hypothesized that the female rats in the

present study would display significantly higher rates of craving than the male rats. In addition,

because of the results of Grimm et al., (2001), which suggested that the amount of craving

increased across the first two months following the initial withdrawal period, it was also

hypothesized that the amount of craving would also increase across both Day 1, Day 18, and

would reach its height at Day 22.

Method

Subjects

Twelve Sprague-Dawley rats were included in the study. Subjects were comprised of 6

male and 6 female adult rats obtained from Envigo (Envigo, Indianapolis, IN). The study was

granted approval from the Institutional Animal Care and Use Committee. Upon review of the
SEX AND INCUBATION OF CRAVING 6

final data, two male rats were eliminated from the study due to their extremely low amount of

active lever presses across the entirety of the study.

Materials

The reinforcers used in the study were 45 mg TestDiet sucrose pellets (TestDiet,

Richmond, IN).

Apparatus

The chambers utilized in the experiment were Graphic State Modular Chambers, Habitest

Model (Coulbourne Instruments LLC, Whitehall, PA). The chambers each contained a house

light on the upper right side of the ceiling, as well as LED lights above the active lever. The

active lever was located in the lower right corner of the right wall of the chamber, and the

inactive lever was located on the left corner of the right wall of the chamber. The software used

was Graphic State IV (Coulbourne Instruments LLC, Whitehall, PA).

Procedure

A 2 x 3, one between-, one within-factor design was used in the study. The independent

variables were sex (male and female) and the abstinence time point (Day 1, Day 18, and Day 22).

The dependent variable of the study was amount of craving, which was measured by the number

of active lever presses recorded during the extinction tests.

The subjects were pair-housed in plastic, shoebox-type cages with free access to food and

water. They were kept on a 12 hr light, 12 hr darkness schedule. Lights came on approximately

at 7 am and were turned off approximately at 7 pm. The subjects were weighed on a triple-

balance beam prior to every session. At the commencement of the experiment, the mean weight

of the male rats was 304.5 mg, while the mean weight of the female rats was 234.83 mg.
SEX AND INCUBATION OF CRAVING 7

Prior to the start of the self-administration period, the subjects completed three

consequative days of shaping in order to familiarize themselves with the food reward system.

Each session lasted 25 min. During this time, the subjects were guided to the active lever and

trained to associate the pressing of the lever with the sugar pellet reward. When the subjects

pressed the active lever and received a food reward, the LED lights would flash and a sound

would play as cues. The inactive lever had no programmed consequences, thus there were no

effects if it were pressed. During shaping, every active lever press resulted in a sugar pellet to be

delivered. Upon completion of the shaping sessions, the subjects advanced into the self-

administration period. Each session during the self-administration period lasted 25 min. At this

point, the researchers began recording the number of active and inactive lever presses each day.

The first three days of the self-administration period were set at fixed ratio of one (FR-1),

meaning that the subjects had to press the active lever one time in order to receive the food

reward. After three days, the subjects advanced onto FR-3, and had to press the active lever

three times in order to receive a sugar pellet. Finally, after three days of FR-3, the subjects

completed three days of FR-5, meaning that they had to press the active lever five times before

they could receive a food reward. On the day following the third FR-5 session, the subjects

completed the first 10 min extinction test, during which no food was rewarded for pressing either

lever. However, the light and sound cues were still triggered by pressing the active lever. Two

more extinction tests were run on Day 18 and Day 22 following the end of the self-

administration period. During the three extinction tests, the researchers recorded the number of

active lever presses by the subjects in order to measure their amount of craving.

Results
The number of inactive and active lever presses was recorded for both the female and the

male subjects across the two week self-administration period (see Figure 1).
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The ANOVA showed a main effect of sex on amount of craving, F(1,8) = 15.506, p = .004,

because the female subjects (M = 64.17) demonstrated a significantly higher amount of craving

than the male subjects (M = 24.42). The ANOVA also revealed a main effect of the abstinence

time point on the amount of craving, F(2,16) = 26.927, p < .001, as there were significant

differences across Day 1, Day 18, and Day 22 (M = 83.29, 24.59, and 25, respectfully). A Post

Hoc with Bonferroni correction revealed a significant difference between Day 1 (M = 83.29) and

both Day 18 and Day 22 (M = 24.59 and 25, respectfully), but no significant difference between

Day 18 and Day 22. Furthermore, the ANOVA showed an interaction effect between sex and the

abstinence time point, F(2,16) = 4.917, p = .022. Thus, both sex and the abstinence time point

appeared to have significant effects on the amount of craving (see Figure 2).

Discussion

Both the sex and the abstinence time point had significant effects on the amount of

craving. There were significant differences in the amount of craving between the male and

female subjects, as well as across the three extinction tests on Day 1, Day 18, and Day 22. There

was also an interaction effect present between the sex of the subjects and the abstinence time

period. Both variables therefore had significant effects on the subjects amount of craving in

regards to the incubation of craving phenomenon.

The current study hypothesized that the female subjects would demonstrate higher levels

of craving than the male rats. This hypothesis was supported by the data, as significant

differences were indeed detected between males and females, with the female rats pressing the

active lever during the extinction tests significantly more times than the male rats across all three

abstinence time points. Additionally, the study also hypothesized that the amount of craving

would increase for both males and females across the three extinction test days, as previously
SEX AND INCUBATION OF CRAVING 9

demonstrated in similar studies in the incubation of craving phenomenon. However, this

hypothesis was actually not supported by the data; in fact, the data actually showed the complete

opposite effect, with the amount of craving significantly decreasing across abstinence time points

for both females and males. This may be attributed to the fact that the self-administration period

was not long enough for the rats to develop any strong dependence to the sugar pellets. It may

also be that perhaps the sugar pellets were not a strong enough reinforcement for the rats, in

comparison to substances such as alcohol and cocaine.

A large difference between the current studies and past studies such as Grimm et al.

(2001), Madsen et al. (2016), and Cooper, Barnea-Ygael, Levy, Shaham, and Zangen (2006) was

the decision of the current study to choose sucrose reinforcement pellets instead of cocaine.

Additionally, the self-administration periods in the studies by Lu, Grimm, Dempsey, and Shaham

(2004) and Madsen et al. (2016) lasted 6 hrs per day, while, in the current study, the self-

administration sessions only lasted 25 min per day. In the study by Lu et al. (2004), the subjects

continued to be exposed to the addictive substance cues for six months following the withdrawal

date, and the abstinence time points were 1 day and 1, 3, and 6 months after withdrawal. The

current study ended after Day 22 following withdrawal. In addition, the study by Reichel et al.

(2012) kept the rats restricted to food prior to the self-administration period, while the current

study allowed for the subjects to have free access to food and water.

A major difference between the study by Klump, Racine, Hildebrandt, and Sisk (2013)

and the current study was that the current study primarily focused on sex differences in regards

to the incubation of craving phenomenon, as opposed to binge eating and food addiction patterns.

Additionally, the studies by Li et al. (2014), Lu et al. (2004), Grimm et al. (2001), and Madsen et

al. (2016) displayed data within their results which supported the incubation of craving
SEX AND INCUBATION OF CRAVING 10

phenomenon of craving increasing across the withdrawal period, while the current study did not

support this at all.

Although further research is needed to completely understand the relationship between

sex and the incubation of craving, the findings of the present study suggest that sex does indeed

have a significant effect on the amount of craving experienced in subjects following the end of a

self-administration period of an addictive substance. Additionally, the current study also

suggests that males and females may react differently to environmental cues of craving during a

withdrawal period. However, the current study does contradict previous findings that craving

increases over time past the initial withdrawal period, and it is unclear as to why this study

demonstrated a significant decrease in craving across all three extinction time points. The results

of this study imply not only that sex differences are crucial in the understanding of addiction and

relapse, but also that the incubation of craving phenomenon may not be more complex than it

appears.
SEX AND INCUBATION OF CRAVING 11

References

Cooper, A., Barnea-Ygael, N., Levy, D., Shaham, Y., & Zangen, A. (2007). A conflict model of

cue-induced relapse to cocaine seeking. Psychopharmacology, 194, 117-125.

Grimm, J. W., Hope, B. T., Wise, R. A., & Shaham, Y. (2001). Incubation of cocaine craving

after withdrawal. Nature, 412(6843), 141142.

Klump. K., Racine, S., Hildebrandt, B., & Sisk, C. (2013). Sex differences in binge eating

patterns in male and female adult rats. International Journal of Eating Disorders, 46,

729-736.

Kosten, T., & Zhang X. (2008). Sex differences in non-reinforced responding for cocaine.

American Journal of Drug & Alcohol Abuse, 34(4), 473-489.

Lu, L., Grimm, J., Dempsey, J., & Shaham, Y. (2004). Cocaine seeking over extended

withdrawal periods in rats: Different time courses of responding included induced by

cocaine cues versus cocaine priming over the first six months. Psychopharmacology, 176,

101-108.

Madsen, H., Zbukvic, I., Luikinga, S., Lawrence, A., & Kim, J. (2016). Extinction of conditioned

cues attenuates incubation of cocaine craving in adolescent and adult rats. Neurobiology

of Learning and Memory.

Peng, L., Ping, W., Xin, X., Fan, Y., Wang, G., Wang, F., Lu, L. (2014). Incubation of

alcohol craving during abstinence in patients with alcohol dependence. Addiction

Biology, 20, 513-522.

Reichel, C., Chan, C., Ghee, S., & See, R. (2012). Sex differences in escalation of

methamphetamine self-administration: cognitive and motivational consequences in rats.

Psychopharmacology, 223, 371-380.

SEX AND INCUBATION OF CRAVING 12

Sanchez, V., Moore, C., Brunzell, D., & Lynch, W. (2014.) Sex differences in the effect of wheel

running on subsequent nicotine-seeking in a rat adolescent-onset self-administration

model. Psychopharmacology, 231(8), 1753-1762.

Wetherill, R., Jagannathan, K., Hager, N., Childress, A., & Franklin, T. (2015). Sex differences

in associations between cannabis craving and neural responses to cannabis cues:

Implications for treatment. Experimental and Clinical Psychopharmacology, 23(4), 238-

246.
SEX AND INCUBATION OF CRAVING 13

500
Male Active Female Active
400
Male Inactive Female Inactive

300
Responses

200

100

-100

Self Administration Days

Figure 1. Figure 1 displays the number of inactive and active lever presses across the entire self-

administration period. The data shown are the mean scores of inactive and active lever presses

from both the female and male subjects. The error bars depict the standard error of the mean for

each group.
SEX AND INCUBATION OF CRAVING 14

160
140 Males Females
Number of Active Presses

120
100
80
60
40
20
0
Day 1 Day 18 Day 22
Abstience Time Point

Figure 2. Figure 2 displays the effect of sex (male and female) and abstinence time point (1, 18,

and 22 days) on the amount of craving. The data shown are the mean scores of each group, and

the error bars depict the standard error of the mean for each group. There was a main effect for

both sex and abstinence time point, as well as an interaction effect. The data were analyzed with

a two-way mixed design ANOVA.