2022 Undergraduate Research Symposium: Majd Yahya Graduate co-author: Cameron Davidson Opioid addiction is a prominent issue that impacts many people across the world. Several studies detail opioid addiction patterns; however, few studies have investigated long-term cognitive effects of fentanyl following its abstinence. This study explores this shortcoming utilizing a chronic escalating dose of fentanyl model. Male and female Wistar rats had three daily subcutaneous injections (3h intertrial-interval) of either saline (control), low- or high-dose fentanyl, over 12 days. Following saline or fentanyl exposure, rats were subjected to a nine-day abstinence period followed by a five-day Barnes maze assay. The Barnes maze is a circular platform containing 20 uniformly spaced holes along its edge, with only one hole containing a hidden box. Latency and distance traveled to enter the box were measured. All animals experienced two trials per day. Day one consisted of an acquisition phase where rats explored the maze and were guided to the hidden zone if they did not enter it themselves. Days two through five utilized aversive stimuli (120-watt light and 80 dB white noise) which were terminated once rats entered the goal box. Results demonstrated that the high-dose fentanyl rats took significantly longer time to reach the hidden zone on day one in comparison to saline- or low-exposed rats, however, this varied by sex. This data suggests that initial exploratory behavior was subtly impacted by our exposure paradigm, but this effect was transitory. Analyses of collected brain tissue assessing neurochemical differences are to be conducted. Future investigations parsing the cognitive impact of synthetic, semi-synthetic, and organic opioids are planned.

1. INTRODUCTION
Chronic Escalating Fentanyl Administration Induces Differences in Initial Cognitive
Response Following Fentanyl Abstinence in Male and Female Rats
Majd Yahya*, Cameron Davidson*,&, Nareen Sadik*,&, Shane Perrine*,&
Department of Psychiatry and Behavioral Neurosciences*, John D. Dingell VA Medical Center&
METHODS
• Opioid addiction is a prominent societal issue that
impacts many people across the world.
• Very few studies have investigated the long-term
cognitive effects of fentanyl, a synthetic opioid that is
gaining societal prominence.
• The utilization of a Barnes Maze behavioral test allows
for the examination of behavioral and cognitive
outcomes, viewing spatial memory.
• We anticipate biological sex differences to be present
among male and female rats after fentanyl abstinence.
• Male and female rats were subjected to three daily
subcutaneous injections (3-hour Intertrial Interval) of
either saline, low-dose fentanyl, or high-dose fentanyl
over a period of 12 days.
• The dose escalated every 3 days, corresponding to:
• After fentanyl exposure, the rats were subjected to a
9-day forced abstinence period in their home cages.
• Following abstinence, rats completed a 5-day Barnes
Maze behavioral test (2 trials per day).
CONCLUSIONS AND FUTURE DIRECTIONS
FEMALES TRAVELED MORE ON THE BARNES MAZE
• All trials were preceded by 30
seconds of subject-restriction in
the center of the maze.
• Day 1 consisted of a
habituation/acquisition phase.
• Days 2 through 5 utilized
aversive stimuli.
• All trials were stopped when the
animals entered the hidden
zone.
• Distance traveled and latency
to the hidden zone were
dependent measures.
• Maze behavior was recorded
using the Noldus Ethovision XT
activity software. Data was
extracted utilizing this program.
• Data was analyzed and
graphed by using GraphPad
Prism Version 9.
NO FENTANYL-INDUCED CHANGE IN DISTANCE TRAVELED BY SEX
• Figure 3A shows the distance traveled on the Barnes maze
by sex regardless of the fentanyl dose.
• Significant interaction Day x Sex and Sidak’s multiple
comparisons test showed significant difference between
male and female rats on both trials for day 1.
FENTANYL INCREASED LATENCY TO HIDDEN ZONE
• Figure 3B and Figure 3C show the distance traveled by the
rats that were exposed to fentanyl, separated by sex.
• As the Barnes Maze proceeded, the rats got better with
time, as they did not have to travel as far prior to reaching
the hidden zone.
• Sex is an important behavioral indicator when viewing initial
behavior differences on a Barnes Maze following fentanyl
exposure, but does not impact overall long-term spatial
memory abilities.
• Training of the Barnes maze behavior was successful by
day 3, and subtle sex-differences were found on day 1,
regardless of fentanyl exposure.
• Future investigations will:
• Utilize a probe trial and/or reversal learning in order to
examine cognitive flexibility following opioid exposure.
• Parse the cognitive impact of the synthetic opioids
(fentanyl) vs semi-synthetic (oxycodone) vs organic
(morphine/heroin).
• Explore polydrug use (cocaine and fentanyl) and its
effects on learning and cognitive flexibility.
Fent Adm
in
D1
Fent Adm
in
D2
Fent Adm
in
D4
Fent Adm
in
D6
Fent Adm
in
D8
Fent Adm
in
D10
Fent Adm
in
D12
Abstinence
Day
3
Abstinence
Day
5
Abstinence
Day
7
Barnes
M
aze
Day
1
Barnes
M
aze
Day
3
Barnes
M
aze
Day
5
250
300
350
400
450
500
Male Weight Data
Day of Barnes Maze
Weight
(in
grams
+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
Fent Adm
in
D1
Fent Adm
in
D2
Fent Adm
in
D4
Fent Adm
in
D6
Fent Adm
in
D8
Fent Adm
in
D10
Fent Adm
in
D12
Abstinence
Day
3
Abstinence
Day
5
Abstinence
Day
7
Barnes
M
aze
Day
1
Barnes
M
aze
Day
3
Barnes
M
aze
Day
5
200
250
300
Female Weight Data
Day of Barnes Maze
Weight
(in
grams
+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
30
60
90
120
Male Fent + Abstinence on Barnes
Maze Latency (All Days)
Day of Barnes Maze
Latency
in
Seconds
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
30
60
90
120
Female Fent + Abstinence on Barnes
Maze Latency (All Days)
Day of Barnes Maze
Latency
in
Seconds
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Distance Traveled by Sex
Trials Separated
DistanceTraveled
(cm)
(+/-
SEM)
FEMALE
MALE
✱✱✱✱✱✱✱✱
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Male Fent + Abstinence on Barnes
Maze Distance Traveled
Day of Barnes Maze
DistanceTraveled
(cm)
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Female Fent + Abstinence on Barnes
Maze Distance Traveled
Day of Barnes Maze
DistanceTraveled
(cm)
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
Spatial learning is unaffected
by fentanyl withdrawal but is
affected by sex.
• Figure 2 illustrates the latency to hidden zone by day and
trial, separated between the sexes and by fentanyl dose.
• Significant interaction between fentanyl dose and Barnes
maze day for both male [F(18,153) = 2.402, p < 0.01] and
female rats [F(18,153) = 1.864, p < 0.05]
• Tukey’s MCT Post hoc procedure for males shows a
significant difference between Fentanyl High and Control
only on Day 1 Trial 1 (p = 0.0213) but all other
comparisons were not significant.
• Tukey’s MCT post hoc procedure for females showed no
significant comparisons.
RAT WEIGHT INCREASED STEADILY
• The weight of both male and female rats steadily
increased, with no differences in observable trends
occurring between the sexes (as shown in figure 1A).
Figure 1A (Male) and 1B (Female) – Rat
Weights
Figure 2A (Male) and 2B (Female) – Latency
to Hidden Zone
Figure 3A – Distance Traveled Collapsed Across Dose
Figure 3B – Male Rats Distance Traveled Separated by Dose Figure 3C – Female Rats Distance Traveled Separated by Dose
20 Hole custom
Built Barnes
Maze
120 cm
Diameter
Barnes Maze Specifications
Representative Heatmap
Showing the Pathway of a Rat
Exposure Period
3 Injections/Day
12 Days
Barnes Maze
2 Trials/Day
5 Days
ACKNOWLEDGEMENTS
• Funding: R01 DA042057/DA/NIDA/NIH HHS/United States
• Fentanyl sourced from: NIDA Control Supply Program
(Bethesda, Maryland).

2. •Opioid addiction is a prominent societal issue that impacts
many people across the world.
•Very few studies have investigated the long-term cognitive
effects of fentanyl, a synthetic opioid that is gaining societal
prominence.
•The utilization of a Barnes Maze behavioral test allows for
the examination of behavioral and cognitive outcomes,
viewing spatial memory.
•We anticipate biological sex differences to be present
among male and female rats after fentanyl abstinence.
INTRODUCTION

3. METHODS
• All rats were subjected to 3 daily
subcutaneous injections (3-hour Intertrial
Interval) of either saline, low-dose fentanyl, or
high-dose fentanyl over a period of 12 days.
• The dose escalated every 3 days,
corresponding to:
Exposure Period
3 Injections/Day
12 Days
Barnes Maze
2 Trials/Day
5 Days
•Barnes Maze Day 1 consisted of a
habituation/acquisition phase, while days 2
through 5 utilized aversive stimuli.
•All trials were stopped when rats entered the
hidden zone. Distance traveled and latency
to the hidden zone were dependent
measures.
•Maze behavior was recorded using the
Noldus Ethovision XT activity software, and
data was analyzed and graphed through
GraphPad Prism Version 9.
20 Hole custom
Built Barnes Maze
120 cm
Diameter
Barnes Maze Specifications
Experimental Timeline:

4. RESULTS: FENTANYL INCREASED LATENCY TO HIDDEN ZONE
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
30
60
90
120
Male Fent + Abstinence on Barnes
Maze Latency (All Days)
Day of Barnes Maze
Latency
in
Seconds
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
30
60
90
120
Female Fent + Abstinence on Barnes
Maze Latency (All Days)
Day of Barnes Maze
Latency
in
Seconds
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
Figure 2A (Male) and 2B (Female) – Latency to Hidden
Zone
•Figure 2 illustrates the latency to
hidden zone by day and trial,
separated between the sexes and
by fentanyl dose.

5. RESULTS: FEMALES TRAVELED MORE ON THE BARNES MAZE
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Distance Traveled by Sex
Trials Separated
DistanceTraveled
(cm)
(+/-
SEM)
FEMALE
MALE
✱✱✱✱✱✱✱✱
Figure 3A – Distance Traveled Collapsed Across Dose

6. RESULTS: NO FENTANYL-INDUCED CHANGE IN DISTANCE TRAVELED BY SEX
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Male Fent + Abstinence on Barnes
Maze Distance Traveled
Day of Barnes Maze
DistanceTraveled
(cm)
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Female Fent + Abstinence on Barnes
Maze Distance Traveled
Day of Barnes Maze
DistanceTraveled
(cm)
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
Figure 3B – Male Rats Distance Traveled
Separated by Dose
Figure 3C – Female Rats Distance Traveled
Separated by Dose

7. •Sex is an important behavioral indicator when viewing initial behavior
differences on a Barnes Maze following fentanyl exposure, but does not impact
overall long-term spatial memory abilities.
•Training of the Barnes maze behavior was successful by day 3, and subtle sex-
differences were found on day 1, regardless of fentanyl exposure.
CONCLUSIONS
FUTURE DIRECTIONS
•Future investigations will:
•Utilize a probe trial and/or reversal learning in order to examine cognitive
flexibility following opioid exposure.
•Parse the cognitive impact of the synthetic opioids (fentanyl) vs semi-synthetic
(oxycodone) vs organic (morphine/heroin).
•Explore polydrug use (cocaine and fentanyl) and its effects on learning and
cognitive flexibility.

8. INTRODUCTION
Chronic Escalating Fentanyl Administration Induces Differences in Initial Cognitive
Response Following Fentanyl Abstinence in Male and Female Rats
Majd Yahya*, Cameron Davidson*,&, Nareen Sadik*,&, Shane Perrine*,&
Department of Psychiatry and Behavioral Neurosciences*, John D. Dingell VA Medical Center&
METHODS
• Opioid addiction is a prominent societal issue that
impacts many people across the world.
• Very few studies have investigated the long-term
cognitive effects of fentanyl, a synthetic opioid that is
gaining societal prominence.
• The utilization of a Barnes Maze behavioral test allows
for the examination of behavioral and cognitive
outcomes, viewing spatial memory.
• We anticipate biological sex differences to be present
among male and female rats after fentanyl abstinence.
• Male and female rats were subjected to three daily
subcutaneous injections (3-hour Intertrial Interval) of
either saline, low-dose fentanyl, or high-dose fentanyl
over a period of 12 days.
• The dose escalated every 3 days, corresponding to:
• After fentanyl exposure, the rats were subjected to a
9-day forced abstinence period in their home cages.
• Following abstinence, rats completed a 5-day Barnes
Maze behavioral test (2 trials per day).
CONCLUSIONS AND FUTURE DIRECTIONS
FEMALES TRAVELED MORE ON THE BARNES MAZE
• All trials were preceded by 30
seconds of subject-restriction in
the center of the maze.
• Day 1 consisted of a
habituation/acquisition phase.
• Days 2 through 5 utilized
aversive stimuli.
• All trials were stopped when the
animals entered the hidden
zone.
• Distance traveled and latency
to the hidden zone were
dependent measures.
• Maze behavior was recorded
using the Noldus Ethovision XT
activity software. Data was
extracted utilizing this program.
• Data was analyzed and
graphed by using GraphPad
Prism Version 9.
NO FENTANYL-INDUCED CHANGE IN DISTANCE TRAVELED BY SEX
• Figure 3A shows the distance traveled on the Barnes maze
by sex regardless of the fentanyl dose.
• Significant interaction Day x Sex and Sidak’s multiple
comparisons test showed significant difference between
male and female rats on both trials for day 1.
FENTANYL INCREASED LATENCY TO HIDDEN ZONE
• Figure 3B and Figure 3C show the distance traveled by the
rats that were exposed to fentanyl, separated by sex.
• As the Barnes Maze proceeded, the rats got better with
time, as they did not have to travel as far prior to reaching
the hidden zone.
• Sex is an important behavioral indicator when viewing initial
behavior differences on a Barnes Maze following fentanyl
exposure, but does not impact overall long-term spatial
memory abilities.
• Training of the Barnes maze behavior was successful by
day 3, and subtle sex-differences were found on day 1,
regardless of fentanyl exposure.
• Future investigations will:
• Utilize a probe trial and/or reversal learning in order to
examine cognitive flexibility following opioid exposure.
• Parse the cognitive impact of the synthetic opioids
(fentanyl) vs semi-synthetic (oxycodone) vs organic
(morphine/heroin).
• Explore polydrug use (cocaine and fentanyl) and its
effects on learning and cognitive flexibility.
Fent Adm
in
D1
Fent Adm
in
D2
Fent Adm
in
D4
Fent Adm
in
D6
Fent Adm
in
D8
Fent Adm
in
D10
Fent Adm
in
D12
Abstinence
Day
3
Abstinence
Day
5
Abstinence
Day
7
Barnes
M
aze
Day
1
Barnes
M
aze
Day
3
Barnes
M
aze
Day
5
250
300
350
400
450
500
Male Weight Data
Day of Barnes Maze
Weight
(in
grams
+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
Fent Adm
in
D1
Fent Adm
in
D2
Fent Adm
in
D4
Fent Adm
in
D6
Fent Adm
in
D8
Fent Adm
in
D10
Fent Adm
in
D12
Abstinence
Day
3
Abstinence
Day
5
Abstinence
Day
7
Barnes
M
aze
Day
1
Barnes
M
aze
Day
3
Barnes
M
aze
Day
5
200
250
300
Female Weight Data
Day of Barnes Maze
Weight
(in
grams
+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
30
60
90
120
Male Fent + Abstinence on Barnes
Maze Latency (All Days)
Day of Barnes Maze
Latency
in
Seconds
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
30
60
90
120
Female Fent + Abstinence on Barnes
Maze Latency (All Days)
Day of Barnes Maze
Latency
in
Seconds
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Distance Traveled by Sex
Trials Separated
DistanceTraveled
(cm)
(+/-
SEM)
FEMALE
MALE
✱✱✱✱✱✱✱✱
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Male Fent + Abstinence on Barnes
Maze Distance Traveled
Day of Barnes Maze
DistanceTraveled
(cm)
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
D1 T1 D1 T2 D2 T1 D2 T2 D3 T1 D3 T2 D4 T1 D4 T2 D5 T1 D5 T2
0
500
1000
1500
2000
2500
Female Fent + Abstinence on Barnes
Maze Distance Traveled
Day of Barnes Maze
DistanceTraveled
(cm)
(+/-
SEM)
Control (Saline)
Fentanyl Low
Fentanyl High
Spatial learning is unaffected
by fentanyl withdrawal but is
affected by sex.
• Figure 2 illustrates the latency to hidden zone by day and
trial, separated between the sexes and by fentanyl dose.
• Significant interaction between fentanyl dose and Barnes
maze day for both male [F(18,153) = 2.402, p < 0.01] and
female rats [F(18,153) = 1.864, p < 0.05]
• Tukey’s MCT Post hoc procedure for males shows a
significant difference between Fentanyl High and Control
only on Day 1 Trial 1 (p = 0.0213) but all other
comparisons were not significant.
• Tukey’s MCT post hoc procedure for females showed no
significant comparisons.
RAT WEIGHT INCREASED STEADILY
• The weight of both male and female rats steadily
increased, with no differences in observable trends
occurring between the sexes (as shown in figure 1A).
Figure 1A (Male) and 1B (Female) – Rat
Weights
Figure 2A (Male) and 2B (Female) – Latency
to Hidden Zone
Figure 3A – Distance Traveled Collapsed Across Dose
Figure 3B – Male Rats Distance Traveled Separated by Dose Figure 3C – Female Rats Distance Traveled Separated by Dose
20 Hole custom
Built Barnes
Maze
120 cm
Diameter
Barnes Maze Specifications
Representative Heatmap
Showing the Pathway of a Rat
Exposure Period
3 Injections/Day
12 Days
Barnes Maze
2 Trials/Day
5 Days
ACKNOWLEDGEMENTS
• Funding: R01 DA042057/DA/NIDA/NIH HHS/United States
• Fentanyl sourced from: NIDA Control Supply Program
(Bethesda, Maryland).