Molly Williams 2 , Nathaniel Stafford 1 and Robert Drugan 1

1. Do ultrasonic vocalizations during intermittent swim stress forecast subsequent alcohol reactivity? Molly Williams2 , Nathaniel Stafford1 and Robert Drugan1 1Psychology Department, University of New Hampshire, Durham, NH 03824, USA 2Neuroscience and Behavior, University of New Hampshire, Durham, NH 03824, USA Materials Apparatus Introduction Conclusions • There was one caller throughout the study. The caller was part of the ISS/Saline group. • The results indicate a significant drug main effect • The results did not indicate a significant effect of stress • Adding more animals to the study would increase the power, and the potential number of callers. Intermittent Swim Stress (ISS) Anxiety disorders affect 18.1% of adults in the United States. Anxiety is a normal response to stress. In abnormal amounts, however, stress can cause disorders such as generalized anxiety disorder, obsessive compulsive disorder, panic disorder, post-traumatic stress disorder and depression. According to the National Institute of Mental Health, only 36.9% of those with an anxiety disorder are receiving treatment and 34.3% of those are receiving a minimally adequate treatment (NIMH 2009). The implications of treatment deficiency demonstrate the need for further investigation of stress related disorders in order to continue to improve quality of life for those suffering from the effects of stress and anxiety. Those who do not receive successful professional treatment often resort to dangerous self-medication with alcohol, as it is often perceived as an effective coping mechanism (Robinson, Sareen, Cox, Bolton 2009). Alcohol is known to be a central nervous system (CNS) depressant, impairing reaction time, balance, and motor coordination through its action on GABA receptors in the CNS. As both alcohol and stress act on a common neurotransmitter site in the brain, GABA-BDZ, subsequent studies have found that stressed rats react more strongly to drugs that act on this site, such as ethanol, when compared to non-stressed controls (Drugan et al., 1992; Austin, Myles, Brown 1999). This suggests that self-medication during stress may result in an increase in the reinforcing effects of alcohol and thereby lead to alcohol abuse and possibly addiction. Previous studies have also investigated the implications of uncontrollable versus controllable shock stress, finding that only those rats that cannot control the stressor will exhibit greater sleep time and motor ataxia as a result of ethanol administration (Drugan et al., 1992; Drugan et al., 1996). Recent evidence in a new swim stress model suggests that resilience can be seen in rats that do not have the opportunity for active behavioral coping, yet emit significant amounts of ultrasonic vocalizations (USV). The ISS model has been shown to result in anxiety and depression (Christianson and Drugan 2005; Warner et al., 2013). Rats that exhibit high amounts of vocalizations will show reduced reactivity to alcohol in comparison to non callers. Subjects 24 male Sprague-Dawley rats weighing 250-300 g were used for the experiment. The rats were housed four per cage in the vivarium with free access to food and water on a 12-hour light/dark cycle. The lights were on at 6:00 am. The rats were randomly assigned to one of four groups: CC/saline, ISS/saline, CC/Ethanol, ISS/ethanol. Drug 20% ethanol was administered intraperitoneally at a dose of 0.6 mg/kg of the animal’s body weight. The control group received an equal volume of 0.9% saline. Rotarod Motor ataxia was measured using the Rotarod treadmill model 7700 . The rotarod has a diameter of 6cm and is 35cm in length and rotates at a constant speed of 10 rotations per minute. (Below) One stress animal is lowered into water by cylinder on the left while a control is simultaneously lowered in the shorter cylinder. Rats were warmed with fans between trials. References Rotarod Austin, M., Myles, V., Brown, P.L, Mammola, B., Drugan, R. (1999). FG 7142- and Rat Induced Alterations in the Ataxic Effects of Alcohol and Midazolam in Rats are Time Dependent. Pharmacology Biochemistry and Behavior, 62, 45-51. Christianson, J.P, Drugan, R. (2005) Intermittent cold water swim stress increases immobility and interferes with escape performance in rat. Behavioral Brain Research, 165(1):58-62 Drugan, R. C, Christianson, J. P, Warner, T. A, Kent, S. (2013). Resilience in shock and swim stress models of depression. Frontiers in behavioral neuroscience. 7(14):1-8. Drugan, R.C, Coyle, T.S, Healy, D.J, Chen, S. (1996). Stress Controllability Influences the Ataxic Properties of Both Ethanol and Midazolam in the Rat. Behavioral Neuroscience, 110 (2), 360-367. Drugan, R.C, Scher D.M, Sarabanchong, V., Guglielmi, A., Meng, I., Chang, J., Bloom, K., Sylvia S., Holmes, P. (1992). Controllability and Duration of Stress Alter Central Nervous System Depressant- Induced Sleep Time in Rats. Behavioral Neuroscience, 106 (4), 682-689. National Institute of Mental Health. 2009. Anxiety Disorder Among Adults. Retrieved from <http://www.nimh.nih.gov/statistics/1ANYANX_ADULT.shtml>. Robinson, J., Sareen, J., Cox, B.J, Bolton, J. 2009. Self-medication of anxiety disorders with alcohol and drugs: Results from a nationally representative sample. Journal of Anxiety Disorders, 23 (1), 38-45. Warner, T.A., Lowry, C.A., Stafford., N.P., & Drugan, R.C. (2013). Intermittent swim stress effects on anxiety behavior. Program No. 128.18. Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2013. Online. (Left) Each animal was trained on the rotarod prior to stress, and then again 2 hours post ISS. Motor ataxia was measured 12 min. post injection Procedure Rotarod Criterion Test • All rats were trained to run on the rotarod. • The success of this task was based on their ability to remain on the rotarod for 2 minutes continuously. • Previous studies with a similar procedure have shown that rats have little difficulty learning this task Intermittent Swim Stress (ISS): • ISS rats were exposed to 80 trials of 5-sec cold water swim. • CC rats were placed in a shorter cylinder in the swim apparatus and underwent the intermittent procedure, but never reaching the water. • One ISS and one CC rat simultaneously experienced the procedure. • Ultrasonic vocalizations were recorded during the session from ISS rats. • ISS rats that emitted USVs were termed callers, while those that did not were referred to as non-callers. • Post Stress Injection and Rotarod: • 100 minutes following the ISS, all rats including controls were given a final 2 minute criterion test on the rotarod approximately 2 hours post ISS. • Each rat was then given an intraperitoneal (into the body cavity) injection of either ethanol (0.6 g/kg) or the equivalent volume of saline. • Twelve minutes following the injection all rats were tested for motor ataxia on the rotarod. • Three successive trials up to 300 seconds each were conducted, and the average was taken for three trials. Ultrasonic Frequency Microphone (Right) conditioner used to record ultrasonic vocalizations. The microphone was positioned just below the ISS Results Effects of Alcohol and Ethanol on Motor Ataxia Acknowledgements All behavioral procedures were reviewed and approved by the University of New Hampshire Institutional Animal Care and Use Committee (IACUC). This project was funded by a grant award provided by the Hamel Center for undergraduate research. Special thanks to India Stribling and Nate Stafford for all of their help throughout this project. Hypothesis Figure 1: The mean time (seconds) spent on the rotarod following ISS exposure and interperitoneal injection. Rotarod testing took place twelve minutes following injection.