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MODELING MECHANISMS OF FEAR AND ANXIETY:
NICOTINE WITHDRAWALAND STRAIN DIFFERENCES
Marissa Applegate, Shruthi Deivasigamani, Conor Driscoll, Sumeet Jain, Sarah McAlister, Jacquelyn Olwell, Ravi Pancholi, Claire Rhee, David Gabriel Rissman, Adam Rosenstein, Madison Taormina, Justin Zhang
Advisors: Dr. Graham Cousens and Zack Vogel
Fear AnxietyDirected at a
stimulusDissipates after
stimulus is removed
Construct: Phasic Fear
General uneasinessLong lastingConstruct: Sustained
Fear
Fear vs. Anxiety
Acoustic Startle Response
Auditory
Input
PNC
Motor Output
FearPhasic FearCentral Nucleus
AnxietySustained FearBed Nucleus of Stria
Terminalis (BNST)
Paradigms of Fear and Anxiety
Lateral Nucleus
Central Nucleus BNST
Acoustic Startle Response
Auditory
Input
PNCMotor Output
Amygdala
Experiment 1: Effects of Strain Differences on the Light Enhanced Startle Response in Lewis and Sprague- Dawley Rats
Experiment 2: Effects of Nicotine Withdrawal on Discrete Cue and Contextual Conditioned Fear
Experiment 3: Nicotine Withdrawal-Induced Anxiety in the Elevated Zero Maze
Road Map
Effects of Strain Differences on the Light Enhanced Startle Response in Lewis and Sprague-Dawley Rats
Evaluate genetic differencesMeasure baseline startle response (1,3)Light-enhanced startle (LES) (2,4,5)Impact on future research
Hypotheses – Lewis vs. Sprague-DawleyGreater baseline startleGreater percent increase of LES startle
responseSustained fear present after LES
Experiment 1
Sprague-Dawley Rat H59-H656 male
Lewis RatH51-588 male
SubjectsExperiment 1
A. Coulbourn Precision Animal Shocker
B. Light C. FanD. Plexiglass cover for inner
chamberE. Odor Emitter
Startle Chamber
F. Amyl AcetateG. Outer ChamberH. SpeakerI. Accelerometer
Experiment 1 +2
Experiment 1Light Enhanced Startle
Five minute Acclimation Period40 pulses at
75, 85, 95, 105 dB30 seconds apart
Light
Five minute Acclimation Period
40 pulses at 75, 85, 95, 105 dB30 seconds apart
Dark
Five minute Acclimation Period40 pulses at
75, 85, 95, 105 dB30 seconds apart
Dark
Five minute Acclimation Period
40 pulses at75, 85, 95, 105 dB30 seconds apart
Dark
Baseline Response
75 85 95 1050
0.00005
0.0001
0.00015
0.0002
0.00025
0.0003
0.00035
0.0004
0.00045
0.0005
LewisSprague-Dawley
Decibel Level
Sta
rtle
Resp
onse
The Lewis rats had a higher baseline startle response when compared to the Sprague-Dawleys.
Experiment 1
0
20
40
60
80
100
120
140
160
180
Lewis
DDD
DLD
2 3
Perc
ent
of
Base
line
0
20
40
60
80
100
120
140
160
Sprague Dawley
DDDDLD
2 3
Perc
ent
of
Base
line
At the 95 dB level, the Sprague Dawley rats exhibited a higher LES startle response, and a greater percent increase in startle. The Sprague-Dawley also exhibited a sustained fear.
Percent Baseline Startle at 95 dB
Experiment 1
0
50
100
150
200
250
Lewis
DDDDLD
2 3
PE
rcent
of
Base
line
0
50
100
150
200
250
Sprague-Dawley
DDDDLD
2 3
At the 105 dB level, the Sprague-Dawley rats still exhibited a higher LES reaction and a greater percent increase.
Percent Baseline Startle at 105 dB
Experiment 1
• 43.5% try to quit smoking
• Of those, between 70% and 90% fail (6)
• Connection between nicotine withdrawal and anxiety
• Treatments to limit withdrawal effects
Why research withdrawal?
Link between withdrawal and sustained fear
Withdrawal and Phasic Fear
HypothesisThere will be no effect on phasic fear.
Effects of Nicotine Withdrawal on Discrete Cue and
Contextual Conditioned Fear
Experiment 2
Pavlovian ConditioningExperiment 2
Subjects
Sprague-Dawley Rat H1, H2, H3, H8, H9
5 maleHigh Dosage of Nicotine
(.5mg/kg)
Sprague-Dawley Rat H4, H11, H12, H13, H14
5 maleLow Dosage of Nicotine
(.25mg/kg)
Sprague-Dawley Rat H5, H6, H7, H10
4 maleControl (Saline)
.25.50 .00
Injected every day for three weeksTested two hours after injection
Experiment 2 + 3
MethodsExperiment 2
Previous ResultsExperiment 2
Conclusion: Robust contextual fear potentiated startle with no difference between groups
0
50
100
150
200
250
300
Percent Potentiation of Contextual Fear
Control
Mea
n P
erce
nt
Pot
enti
atio
n
Low Dose High DosePre-conditioning Baseline Context Re-exposure
0
200
400
600
800
1000
1200
1400
Contextual Fear in Sprague-Dawley Rats
Control Low DoseHigh Dose
Mea
n S
tart
le A
mp
litu
de
ResultsExperiment 2
Conclusion: No olfactory fear potentiated startle in all groups
Odor Odor+30 Odor+60 Odor+900
200
400
600
800
1000
1200
1400
Olfactory Fear Conditioning in Sprague-Dawley Rats
ControlLow DoseHigh Dose
Mea
n S
tart
le A
mp
litu
de
-20
-15
-10
-5
0
5
10
Olfactory Fear Potentiation
Control
Per
cen
t P
oten
tiat
ion
Low Dose High Dose
ResultsExperiment 2
Elevated zero-mazeNicotine withdrawal
leads to anxiety
Nicotine Withdrawal-Induced
Anxiety in the Elevated Zero Maze
Hypothesis: As nicotine dosage increases and causes a higher withdrawal affect, anxiety, locomotor activity, and time spent in the closed sections of the maze will increase.
Experiment 3
Zero Maze
Walled
LocomotorActivity
Time Spent in
Open and
Closed Areas
Experiment 3
Nicotine Withdrawal-Induced Anxiety in the Elevated Zero
Maze
Rat placed in maze
15 minute testing period
Nicotine Injections
2 hour waiting period
Experiment 3
0.0 mg/kg 0.25 mg/kg 0.5 mg/kg0
20
40
60
80
100
120
140
160
180
200
Line Crosses vs. Dosage
Dosage of Nicotine
Lin
e C
rosses
0.0 mg/kg 0.25 mg/kg 0.5 mg/kg280
290
300
310
320
330
340
350
360
370
Time Spent in Open vs Dosage
Dosage of Nicotine
Tim
e S
pen
t in
Op
en
(S
eco
nd
s)
Results
Nicotine dosage had no effect on the amount of time the rats spent in the open sections of the maze.
Nicotine dosage had no effect locomotor activity.
Experiment 3
10:00 10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00 14:30 15:00 15:30 16:00 16:30 17:000
50
100
150
200
250
300
350
400
450
500
Time of Day
Tim
e S
pen
t in
Op
en
(se
con
ds)
Time in Open vs. Time of Day
Experiment 3
Surprising Data Need more conclusive dataFuture experiments
Conclusion
AcknowledgementsDr. Graham Cousens , who is getting married today!Dr. Miyamoto, our fearless leaderMyrna Papier, our indispensible organizerZack Vogel, our epic counselor All the NJGSS StaffAnd our gracious benefactors:
John and Laura Overdeck Bayer HealthcareThe Crimmins Family Charitable FoundationNJGSS Alumni and Parents 1984 – 2011Bristol-Meyer SquibbRocheKinder MorganBain Capital Childrens CharityGlastoSmithKlineNovartis
Reference Sources 1. Ramos A, Kangerski AL, Basso PF, Da Silva Santos JE, Assreuy J, Vendruscolo LF, Takahashi RN.
Evaluation of Lewis and SHR rat strains as a genetic model for the study of anxiety and pain. Behavioral Brain Research. 2002 Feb; [Internet]. [cited 2011 Jul 27] 129(1-2): 113-123. Available from: http://www.ncbi.nlm.nih.gov.ezproxy.drew.edu/pubmed/11809502
2. Steiner MA, Lecourt H, Rakotoariniaina A, Jenck F. Favoured genetic background for testing anxiolytics in the fear-potentiated and light-enhanced startle paradigms in the rat. Behavioural Brain Research. 2011 Aug; [Internet]. [cited 2011 Jul 27] 221(1): 34-42. Available from: http://www.ncbi.nlm.nih.gov.ezproxy.drew.edu/pubmed?term=Favoured%20genetic%20background%20for%20testing%20anxiolytics%20in%20the%20fear-potentiated%20and
3. Freet CS, Tesche JD, Tompers DM, Riegel KE, Grigson PS. Lewis rats are more sensitive than Fischer rats to successive negative contrast, but less sensitive to the anxiolytic and appetite-stimulating effects of chlordiazepoxide. Pharmacology, Biochemistry, and Behavior. 2006 Oct; [Internet]. [cited 2011 Jul 27] 85(2): 378-384. Available from: http://www.ncbi.nlm.nih.gov.ezproxy.drew.edu/pubmed/17049372
4. Davis M, Walker DL, Miles L, Grillon C. Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety. Neuropsychopharmacology. 2010 Jan; [Internet]. [cited 2011 Jul 27] 35(1): 105-135. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19693004
5. Jonkman S, Risbrough VB, Geyer MA, Markou A. Spontaneous nicotine withdrawal potentiates the effects of stress in rats. Neuropsychopharmacology. 2008 Aug; [Internet]. [cited 2011 Jul 27] 33(9): 2131-2138. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18033237
6. (Bronars C, Saul J. Increasing Reach of Tobacco Cessation Quitlines: A Review of the Literature and Promising Practices [home page on the Internet]. Phoenix (AZ): North American Quitline Consortium; 2009. [Introduction; cited 2011 July 27]. 18 p. Available from: http://www.naquitline.org/resource/resmgr/issue_papers....)