Beruflich Dokumente
Kultur Dokumente
Meagan E. Malesic
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
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
In regards to sex differences within addiction, Reichel, Chan, Ghee, and See (2012)
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,
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
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
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
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
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).
SEX AND INCUBATION OF CRAVING 8
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
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
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
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
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
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
Grimm, J. W., Hope, B. T., Wise, R. A., & Shaham, Y. (2001). Incubation of cocaine craving
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.
Lu, L., Grimm, J., Dempsey, J., & Shaham, Y. (2004). Cocaine seeking over extended
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
Peng, L., Ping, W., Xin, X., Fan, Y., Wang, G., Wang, F., Lu, L. (2014). Incubation of
Reichel, C., Chan, C., Ghee, S., & See, R. (2012). Sex differences in escalation of
Sanchez, V., Moore, C., Brunzell, D., & Lynch, W. (2014.) Sex differences in the effect of wheel
Wetherill, R., Jagannathan, K., Hager, N., Childress, A., & Franklin, T. (2015). Sex differences
246.
SEX AND INCUBATION OF CRAVING 13
500
Male Active Female Active
400
Male Inactive Female Inactive
300
Responses
200
100
-100
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