MODELING MECHANISMS OF FEAR AND ANXIETY: NICOTINE WITHDRAWAL AND 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 vs. Anxiety Fear Anxiety Directed at a stimulus Dissipates after stimulus is removed Construct: Phasic Fear General uneasiness Long lasting Construct: Sustained Fear

Acoustic Startle Response Auditory Input PNC Change order of animations Motor Output

Paradigms of Fear and Anxiety Phasic Fear Central Nucleus Anxiety Sustained Fear Bed Nucleus of Stria Terminalis (BNST)

Acoustic Startle Response Amygdala Lateral Nucleus Central Nucleus BNST Get rid of cochlear and spinal. Text smaller Put it in an oval PNC Input to PNC to Motor Output Motor Output Auditory Input PNC

Road Map 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 MAKE MORE EXCITING!!!!!!

Effects of Strain Differences on the Light Enhanced Experiment 1 Effects of Strain Differences on the Light Enhanced Startle Response in Lewis and Sprague-Dawley Rats Evaluate genetic differences Measure baseline startle response (1,3) Light-enhanced startle (LES) (2,4,5) Impact on future research Hypotheses – Lewis vs. Sprague-Dawley Greater baseline startle Greater percent increase of LES startle response Sustained fear present after LES Change last hypothesis Or prove it.

Subjects Experiment 1 Lewis Rat H51-58 8 male Sprague-Dawley Rat H59-H65 6 male

Startle Chamber Experiment 1 +2 A. Coulbourn Precision Animal Shocker RELABEL A. Coulbourn Precision Animal Shocker B. Light C. Fan D. Plexiglass cover for inner chamber E. Odor Emitter F. Amyl Acetate G. Outer Chamber H. Speaker I. Accelerometer

Five minute Acclimation Period 40 pulses at 75, 85, 95, 105 dB 30 seconds apart Dark Experiment 1 Light Enhanced Startle Five minute Acclimation Period 40 pulses at 75, 85, 95, 105 dB 30 seconds apart Dark Five minute Acclimation Period 40 pulses at 75, 85, 95, 105 dB 30 seconds apart Light Five minute Acclimation Period 40 pulses at 75, 85, 95, 105 dB 30 seconds apart Dark

Baseline Response Experiment 1 The Lewis rats had a higher baseline startle response when compared to the Sprague-Dawleys.

Percent Baseline Startle at 95 dB Experiment 1 Percent Baseline Startle at 95 dB 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 105 dB Experiment 1 Percent Baseline Startle at 105 dB At the 105 dB level, the Sprague-Dawley rats still exhibited a higher LES reaction and a greater percent increase.

Why research withdrawal? 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

Effects of Nicotine Withdrawal on Contextual Conditioned Fear Experiment 2 Effects of Nicotine Withdrawal on Discrete Cue and Contextual Conditioned Fear Link between withdrawal and sustained fear Withdrawal and Phasic Fear Hypothesis There will be no effect on phasic fear.

Pavlovian Conditioning Experiment 2 Pavlovian Conditioning

Subjects Experiment 2 + 3 .50 .25 .00 Sprague-Dawley Rat H1, H2, H3, H8, H9 5 male High Dosage of Nicotine (.5mg/kg) Sprague-Dawley Rat H4, H11, H12, H13, H14 5 male Low Dosage of Nicotine (.25mg/kg) Sprague-Dawley Rat H5, H6, H7, H10 4 male Control (Saline) .50 .25 .00 Injected every day for three weeks Tested two hours after injection

Experiment 2 Methods

Experiment 2 Previous Results

Experiment 2 Results Conclusion: Robust contextual fear potentiated startle with no difference between groups

Experiment 2 Results Conclusion: No olfactory fear potentiated startle in all groups

Nicotine Withdrawal-Induced Anxiety in the Elevated Zero Maze Experiment 3 Nicotine Withdrawal-Induced Anxiety in the Elevated Zero Maze Elevated zero-maze Nicotine withdrawal leads to anxiety Elevated zero-maze because rats are afraid of heights; control rats = less anxious… more time in open sections of zero-maze; as anxiety increases, locomotor activity decreases because the rats would be averse to any environmental changes 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.

Zero Maze Locomotor Activity Experiment 3 Time Spent in Open and Closed Areas Walled Locomotor Activity

Nicotine Withdrawal-Induced Anxiety in the Elevated Zero Maze Experiment 3 Nicotine Withdrawal-Induced Anxiety in the Elevated Zero Maze Nicotine Injections 2 hour waiting period 15 minute testing period Rat placed in maze

Experiment 3 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.

Time in Open vs. Time of Day Experiment 3 Time in Open vs. Time of Day

Conclusion Surprising Data Need more conclusive data Future experiments

Acknowledgements Dr. Graham Cousens , who is getting married today! Dr. Miyamoto, our fearless leader Myrna Papier, our indispensible organizer Zack Vogel, our epic counselor All the NJGSS Staff And our gracious benefactors: John and Laura Overdeck Bayer Healthcare The Crimmins Family Charitable Foundation NJGSS Alumni and Parents 1984 – 2011 Bristol-Meyer Squibb Roche Kinder Morgan Bain Capital Childrens Charity GlastoSmithKline Novartis

Reference Sources Laura Overdeck Bayer 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....) Laura Overdeck Bayer