Marissa Applegate, Shruthi Deivasigamani, Conor Driscoll, Sumeet Jain, Sarah McAlister, Jacquelyn...

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....)