1. Blocking frustrating memories using the protein synthesis inhibitor cycloheximide and the β- adrenergic receptor antagonist propranolol in an appetitive alternative to learned helplessness Joshua Cohen and Gretchen Hanson Gotthard Muhlenberg College Allentown, PA 18104 Introduction Animals exposed to inescapable shocks and later tested for escape-avoidance perform worse than other animals that are exposed to escapable shocks or are never shocked (Overmier & Seligman, 1967). This phenomenon has been labeled learned helplessness. Much research attempted to create this effect using appetitive paradigms; however, most were unsuccessful (Job, 1988). It has been shown that the formation of new memories depends on de novo protein synthesis (Kandel, 2001). Further, amnesia for new learning can be produced by inhibiting or disrupting this protein synthesis (Riccio et al., 2006). Two drugs that have been shown to produce this effect are the protein synthesis inhibitor cycloheximide and the β-adrenergic receptor antagonist propranolol (Flint et al., 2007; Cahill et al., 2000). The present study employed an appetitive paradigm to create learned helplessness in which rats were exposed to a frustrating experience. Cycloheximide or propranolol were administered prior to frustration trials in an attempt to block the frustrating memory, and therefore, prevent learned helplessness from developing. Results All rats learned to find the reward to an equal extent. There were no behavioral differences during the frustration trial between the groups. Independent t-tests revealed the control group took longer to find the reward after the frustrating experience [Before: M=10.58, SEM=2.38; After: M=23.11, SEM=5.92; t(13)= -1.968, p=.035], while there were no differences between the latencies in the cycloheximide [Before: M=5.59, SEM=.55; After: M=6.55, SEM=1.20; t(14)= -.727, p=.479], or propranolol groups [Before: M=6.99, SEM=2.32; After: M=10.37, SEM=1.78; t(17)= - 1.179, p=.255] (see Figure 1). Control rats took longer to find the reward (CHX: M=6.55, SE=1.20; Propranolol: M=10.37, SE=1.78; Control: M=23.11, SE=5.92) [F (2,29)=5.559, p=.009] and traveled farther to the reward (CHX: M=66.58, SE=12.69; Propranolol: M=94.59, SE=15.48; Control: M=170.16, SE=39.59) [F (2,25)=4.627, p=.020] on the test when compared to the cycloheximide and propranolol groups. An ANOVA showed that control rats took longer to find the buried reward on the last day of training in the spatial memory task [CHX: M=94.67, SEM=23.87; Propranolol: M=87.30, SEM=23.74; Control: M=190.05, SEM=34.22; F(2,29)=4.243, p=.024] (see Figure 2). Method Subjects Ninety-day old, male Long-Evans rats (N=32) were reduced to and maintained at 85% of their free-feeding weights one week prior to and during experimentation. Water was available ad libitum. Rats were maintained on a 12-hour light/dark cycle. Drugs DL-Propranolol hydrochloride ((±)-1-isopropylamino-3-(1-napthyloxy)-2- propranol hydro-chloride) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and prepared at a concentration of 25mg/ml. Cycloheximide (3-[2-(3,5- Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide) was obtained from Sigma-Aldrich (St. Louis, MO, USA) in a solution of concentration 1mg/mL. Rats received 1 mg/kg intraperitoneal (I.P.) injections of either propranolol, cycloheximide, or vehicle (distilled water). Apparatus All training and testing was performed in the Sand Maze (Gotthard, 200?). The Sand Maze consisted of a circular pool 110 cm in diameter filled with sand and elevated 75 cm above the ground. All trials were recorded using the Smart Jr. video tracking system (Harvard Apparatus). Procedure All rats were trained for four days to eat sweetened cereal (Tootie Fruities) out of a container located in the SE quadrant of the Sand Maze. On the fifth day all rats received an injection of cycloheximide (n=11), propranolol (N=10), or distilled water (N=10) thirty minutes prior to the frustrating experience. Each rat received three frustration trials in the Sand Maze, which consisted of being placed in the maze with the container closed, but still containing cereal (holes in the container top allowed the rats to smell and see the cereal). Each trial lasted 3 minutes or until the rat showed no interaction with the container for 60 consecutive seconds. One day later, the rats were put back into the maze with the container opened and the reward available. The latency to begin eating the cereal and the distance traveled before beginning to eat was measured for each trial. Two weeks later, all rats were trained on a spatial version of the Sand Maze. Reward was present on top of the sand on the first day, partially buried on the second, and completely buried on the third. Latency to begin eating the cereal was recorded for each trial. Rats received a probe trial on the fourth day (i.e., no buried reward) and quadrant preference was measured. Discussion The present study showed that frustration could be used to produce learned helplessness in rats and that treatment with cycloheximide or propranolol prior to a frustrating experience prevented the development of learned helplessness. Learned helplessness developed in rats that did not receive cycloheximide or propranolol before frustration, as shown by longer latencies to retrieve reward on the test and by learning deficits in the sand maze. Deficits in memory for the cycloheximide group cannot be attributed to performance effects (e.g., nausea or motoric impairment). However, propranolol has anxiolytic properties that may have contributed to a lack of learned helplessness in this group, rather than a distinct effect on memory. References Cahill, L., Pham, C.A., Setlow, B. (2000). Impaired memory consolidation in rats produced with β- adrenergic blockade. Neurobiology of Learning and Memory, 74, 259-266. Flint, R.W., Valentine, S., Papandrea, D. Jr (2007). Reconsolidation of a long-term spatial memory is impaired by cycloheximide when reactivated with a contextual latent learning trial in male and female rats. Journal of Neuroscience, 148, 823-844. Job, R.F.S. (1988). Interference and facilitation produced by non-contingent reinforcement in the appetitive situation. Animal Learning & Behavior, 16(4), 451-460. Kandel, E. (2001). The molecular biology of memory storage: A dialogue between genes and synapses. Science, 294, 1030-1038. Overmier, J.B. & Seligman, M.E.P. (1967). Effects of inescapable shock upon subsequent escape and avoidance responding. Journal of Comparative and Physiological Psychology, 63, 28-33. Riccio, D.C., Millin, P.M. & Bogart, A.R. (2006). Reconsolidation: A brief history, a retrieval view, and some recent issues. Learning & Memory, 13, 536-544. Day 2: Training Reward buried shallow Days 1-4: Training Container open reward obtainable Day 5: Frustration Injected with either CHX, Propranolol, or Distilled Water 30 min prior to trial Container closed, reward unobtainable Day 6: Test Container open reward obtainable (Two Weeks Later) Day 1: Training Reward slightly exposed Day 3: Training Reward buried Day 4: Test No reward quadrant preference test (Two-week delay) ACKNOWLEDGEMENTS This research was supported in part through a Student Summer Research Grant (to Joshua Cohen) from the office of the Dean of Academic Life at Muhlenberg College.