1. Chronic Nicotine Exposure and Withdrawal Increases Acoustic Startle Response in Mice
Kenneth A. Bisson, Nicole L. Yohn, Julie A. Blendy, Ph.D
University of Pennsylvania, School of Arts and Sciences, Biological Basis of Behavior
INTRODUCTION SUMMARYPROCEDURE
RESULTS
METHOD
REFERENCES
ACKNOWLEDGMENTS
Cigarette use is the number one cause of death and disability in the
United States, resulting in 443,000 or 20% of all deaths per year (U.S.
Department of Health and Human Services, 2010). The low success rate
of abstinence and the strong and strong reinforcing properties of nicotine
often lead tobacco users to cycle between periods of tobacco use and
periods of withdrawal during an attempt to quit.
Previous studies have examined the role of repeated nicotine
exposure and withdrawal to understand that the reward saliency of
nicotine increases after each abstinence period (Hilario, Turner, and
Blendy, 2012). Although reward saliency increases after each
administration and withdrawal period, the role of stress in repeated
nicotine relapse is unknown.
The acoustic startle response (ASR) is a twitch-like motor reflex in
response to an unexpected auditory stimulus (Koch, 1999). ASR is a
powerful behavior assay because it can quantify increased HPA activity in
both human and animal subjects (Dirks et al., 2002; Calhoun et al., 2011).
Specifically, increased levels of CRF augments acoustic startle amplitudes
(Risbrough, Hauger, Pelleymounter, and Geyer, 2003).
The following study utilizes ASR to determine the stress sensitivity of
mice during repeated exposure and withdrawal from nicotine.
Furthermore, role of CRF receptor 1 (CRFR1) in mediating this behavior
was investigated using a CRFR1 antagonist, antalarmin.
Animals
Male C57BL6/J and A112G wild type mice (n=17) were used in this study.
Mice were kept on a 12hr light/dark cycle and all experimental testing was
performed between 9:00am and 5:00pm. All mice were acclimated to the
behavior suite for one hour prior to testing.
Drug Administration
Mice were implanted with an osmotic minipump containing 18mg/kg/day
of nicotine.
Mice were injected (i.p.) with antalarmin (10mg/kg). Antalarmin was
dissolved in 10% dimethyl sulfoxide.
Acoustic Startle Apparatus
Four identical acoustic startle chambers (SR-Labs; San Diego Instruments,
San Diego) were used in this study. Broadband acoustic startle pips were
emitted from a high frequency speaker fixed atop the acoustic startle
chamber and acoustic startle amplitudes were quantified using a
piezoelectric pickup.
Acoustic Startle Procedure
Mice habituated to the acoustic startle chamber for five minutes with 67dB
white noise background. After habituation, 15 rounds of 3 pseudorandom
pips (95, 105, and 115dB SPL) were presented. Interstimulus intervals were
randomly generated at 30 seconds with 2 second steps (26-34 seconds).
Startle responses were recorded as the average voltage emitted per 1ms for
100ms.
Experimental Procedure
On day 0 of the experiment, all mice underwent a baseline session. During
nicotine exposure, all mice were challenged with an ASR session after 12 days
(12D). During the first withdrawal period, mice underwent an ASR session at
24hrs after withdrawal (24hrWD). During the second and third withdrawal
periods, mice underwent an ASR session at the 24hr and 120hr (120hrWD)
withdrawal points. 144 hours after the third withdrawal, mice were
counterbalanced by acoustic startle amplitudes and given either antalarmin
or vehicle. All data was analyzed within tone and between trials of the same
period (e.g. 12D1x12D2x12D3)
Nicotine dependence and the state of withdrawal need to be better
understood to help facilitate abstinence in tobacco smokers.
Specifically, the role of repeated exposures and withdrawals requires
more investigation. This study observed stress phenotypes during
repeated nicotine exposure and withdrawal and also implemented a
CRFR1 antagonist, antalarmin to investigate the biological basis of
stress sensitivity during nicotine administration and withdrawal.
Findings:
• Repeated nicotine exposure and withdrawal increased acoustic
startle amplitudes while mice were on nicotine during the third
trial
• Repeated nicotine exposure and withdrawal increased acoustic
startle at 95dB and 105dB for second and third trials
• A trend of increased acoustic startle is observed at all three
startle tones.
• Antalarmin administration showed a trending decrease in
startle amplitude at 95dB compared to controls. At 105dB, a
trending increase was observed. No trends were observed at
115dB.
Calhoun, P. S., Wagner, H. R., McClernon, F. J., Lee, S., Dennis, M. F., Vrana, S. R., Clancy, C. P., Collie, C. F.,
Johnson, Y. C. & Beckham, J. C. (2011). The effect of nicotine and trauma context on acoustic startle in
smokers with and without posttraumatic stress disorder.
Psychoparmacology, 215(2), 379 – 389.
Dirks, A., Groenink, L., Schipholt, M. I., van der Gugten, J., Hijzen, T. H., Geyer, M. A. & Olivier, B. (2002).
Reduced startle reactivity and plasticity in transgenic mice overexpressing corticotropin-releasing
hormone. Biological Psychiatry, 2002 (51), 583-590.
Hilario, M. R. F., Turner, J. R. & Blendy, J. A. (2012). Reward sensitization: Effects of repeated nicotine
exposure and withdrawal in mice. Neuropsychopharmacology, 37(2010), 2661 – 2670.
Koch, M. (1999). The neurobiology of startle. Progress in neurobiology, 59(1999), 107-128.
Risbrough, V. B., Hauger, R. L., Pelleymounter, M. A. & Geyer, M. A. (2003). Role of corticotropin release
factor (CRF) receptors 1 and 2 in CRF-potentiated acoustic startle in mice. Psyschopharmacology,
17(2003), 178 – 187.
U.S. Department of Health and Human Services. How Tobacco Smoke Causes Disease: The Biology and
Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta, GA: U.S.
Department of Health and Human Services, Centers for Disease Control and Prevention, National Center
for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2010.
Walker, D. L., Miles, L. A. & Davis, M. (2009). Selective participation of the bed nucleus of the stria
terminalis and CRT in sustained anxiety-like versus phasic fear-like responses. Program for
Neuropsychologicalpharmacology and Biological Psychiatry, 33(8), 1291 – 1308.
I would to thank Nicole Yohn for all of her assistance in developing and
performing this experiment. I would also like to thank Dr. Julie Blendy for
all of her support throughout this project and mentoring my research
credit. Research supported by R01 DA033646. The authors declare no
conflict of interest.
AMYGDALA INNERVATION OF ASR
Adapted from Walker (2009) and Koch (1999)
12 DAY NICOTINE
EXPOSURE ASR
24 HOUR WITHDRAWAL
ASR
120 HOUR WITHDRAWAL
ASR
ANTALARMIN
CHALLENGE
* = p<.05, ** = p<.01, # = p<.05 compared to trial 1.
CONCLUSION
These data suggest that repeated nicotine exposure and withdrawal
result in increased stress phenotypes as observed with ASR. CRF-
potentiated ASR has been shown to increase startle at lower decibels
(Dirks et al., 2002), which may explain the downward trend during the
antlamarin trial at 95dB. Further studies need to investigate brain
tissue for the role of CRF in repeated nicotine exposure and
withdrawal.
105dB 105dB 105dB 105dB
CRF
115dB 115dB 115dB 115dB
95dB 95dB 95dB 95dB