Effect of Cycloheximide on Extinction of Odor Discrimination Learning in Rats Alexandra Knoppel, Katherine Janson, Sonnett White, and Gretchen Hanson Gotthard Randolph College Lynchburg, VA Introduction Much research has shown that the administration of protein synthesis inhibitors blocks the formation of new fear memories (e.g., Nader, Schafe, & LeDoux, 2000). In fact, most of the research in this area has focused on the acquisition of fear responses or used tasks that required animals to respond under aversive conditions (e.g., Morris water maze; Meiri & Rosenblum, 1998). Additionally, most studies examining the effects of blocked protein synthesis have examined acquisition of a completely new response, rather than extinction of an already-established response. A small number of studies have begun to examine the effects of protein synthesis inhibition on extinction (Lattal & Abel, 2001; Suzuki, et al., 2004); however, the results have been mixed and have been conducted with aversive tasks only (e.g., fear conditioning and the water maze). The present studied used an appetitive odor discrimination digging task (Bunsey & Eichenbaum, 1996) to examine the effects of a protein synthesis inhibitor (cycloheximide) on extinction in rats. Considering extinction is similar to acquisition in that it also involves new learning, it was hypothesized that protein synthesis inhibition would block extinction and produce continued high levels of responding during testing. Results Acquisition There were no differences between the Cycloheximide group and the Vehicle group during acquisition [t(9)=.208, p=.84]. Choice performance during acquisition was statistically greater than could be due to chance [M=90.90, SD=12.61; t(10)=10.757, p<.001]. Therefore, all rats included in the present analyses met acquisition criterion (i.e., at least 75% correct overall during training). Extinction All rats met the extinction criterion in three or less extinction trials (i.e., no digging for 30 consecutive seconds). Testing There were no differences in preference scores for the Cycloheximide group (Test 1: M=.37, SD=.50; Test 2: M=.33, SD=.52) and the Vehicle group (Test 1: M=.00, SD=.00; Test 2: M=.20, SD=.45) on Test 1: t(9)=1.636, p=.136, or Test 2: t(9)=.452, p=.662 (see Figure 2). The Cycloheximide group [Test 1: t(5)=1.808, p=.13; Test 2: t(5)=1.581, p=.175] and the Vehicle group [Test 2: t(4)=1.00, p=.374] did not differ from chance on all test trials (see Figure 2). Shaping (Day 1) One cup of unscented sand (two trials) Shaping (Day 2) Two cups of unscented sand (two trials) Shaping (Day 3) Two cups of unscented sand (six trials) Training (Day 4) Trained on one odor discrimination (at least six trials with 75% correct total) Extinction (Day 5) Extinction trials for the odor discrimination (at least two trials) Testing (Day 6) Two test trials for odor discrimination Figure 1

Method Subjects Ninety-day old, male Long-Evans rats (N=11) 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. Apparatus All shaping, training, and testing took place in the rats’ home cages. Plastic Nalgene® cups (125 ml size) were used for the odor discriminations and were mounted using Velcro® onto rectangular Plexiglas® bases. Odor discriminations were created by mixing play sand (148.5 grams) with different dried spices (i.e., 1.5 grams of cocoa or cinnamon). Procedure Rats were shaped to dig in unscented cups of sand for three days prior to training (10 shaping trials total – see Figure 1 for shaping, training, and testing procedure). Rats received one day of training during which they learned one odor discrimination. One day following training, rats received at least two extinction trials for the odor discrimination with a 1 mg/kg cycloheximide injection (n=7) or a vehicle injection (n=6). Twenty-four hours later, rats received two test trials. Discussion Rats learned to dig in the correct odor and showed a statistically significant preference for it in both groups during acquisition. Administration of a protein synthesis inhibitor (cycloheximide) during extinction did not block memory for extinction (as indicated by preference scores that did not differ from chance during testing). Results suggest that protein synthesis may not be critical for the new learning associated with extinction in an appetitive odor discrimination task. References Bunsey, M. & Eichenbaum, H. (1996). Conservation of hippocampal memory function in rats and humans. Nature, 379, Lattal, K. M. & Abel, T. (2001). Different requirements for protein synthesis in acquisition and extinction of spatial preferences and context- evoked fear. The Journal of Neuroscience, 21(15), Meiri, N. & Rosenblum, K. (1998). Lateral ventricle injection of the protein synthesis inhibitor anisomycin impairs long-term memory in a spatial memory task. Brain Research, 789, Nader, K., Schafe, G. E., & LeDoux, J. E. (2000). Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature, 406, Suzuki, A., Josselyn, S. A., Frankland, P. W., Masushige, S., Silva, A. J., & Kida, S. (2004). Memory reconsolidation and extinction have distinct temporal and biochemical signatures. The Journal of Neuroscience, 24(20),