Examination of the State Dependent Properties of WIN-55-212-2 on Spatial Learning and Memory in Rats in the Sand Maze   Ashley R. Smith and Gretchen Hanson Gotthard Randolph-Macon Woman’s College Lynchburg, VA 24503 Results No differences in latency were exhibited between groups on Day 1; however, differences emerged on the first day of training when rats received WIN-2. Overall quadrant preference did not differ between groups, but a breakdown of quadrant preference by minutes revealed poorer performance in the different state group than in the same state groups. Acquisition As can be seen in Figure 1, latency did not differ between groups on Day 1: Shaping [Trial 1, F(2,14) = 0.150, p > .05, and Trial 2, F(2,14) = 1.146, p > .05]. Rats only differed on the first day of drug administration [Trial 3, F(2,14) = 3.563, p = .05]. That is, rats receiving WIN-2 showed longer latencies to find the reward than rats receiving vehicle injections on the first training trial. Quadrant Preference As can be seen in Figure 2, there were no differences in overall quadrant preference between the groups [ F(2,14) = 1.239, p > .05]. However, as seen in Figure 3, a breakdown of quadrant preference by minutes showed significant differences between the groups during Minute 1 [ F(2,14) = 4.794, p < .05] and Minute 4 [F(2,14) = 4.231, p < .05]. An LSD post hoc analysis showed that the different state group spent significantly less time (p < .05) in the correct quadrant than the same state groups. The different state group showed a quadrant preference that was significantly below chance during Minute 4 [t(5) = 3.043, p < .05] and Minute 10 [t(5) = -2.632, p < .05] of the probe trial which would indicate a place aversion to the correct quadrant. Introduction Naturally occurring cannabinoids (e.g., THC) and synthetic cannabinoids (e.g., WIN-55-212-2), have been linked with hippocampal function, and therefore, spatial learning and memory. Although a number of studies have shown a relationship between cannabinoid administration and learning and memory deficits (e.g., Lichtman, et al., 1995), no studies have explicitly examined the state dependent properties of synthetic cannabinoids. State dependent retention refers to the tendency of organisms to recall information better when in the same “state” they were in during learning, than if they were in a different state during learning and testing (Spear and Riccio, 1994). In fact, if cannabinoid administration produces state dependent effects, then a memory impairment following cannabinoid administration may not be due to decreased hippocampal function alone, but may be due to an altered state between acquisition and training or testing. The critical test for state dependent retention is to return the subject to the original state to determine if an inaccessible memory can be retrieved. The sand maze was used in the present study, and is an appetitive open-field spatial task that may serve as an alternative to the water maze in some studies. While the water maze requires rats to swim in a pool of water to locate a hidden platform (Morris, 1981), the sand maze requires rats to dig in a pool of sand to retrieve buried cereal rewards (Hanson, 2003). The sand maze may be a better alternative for studies that aim to examine spatial behavior without the potential side effects of aversive tasks (e.g., increased amygdala activity and/or fight or flight responses). The state dependent properties of the synthetic cannabinoid WIN-55-212-2 (WIN-2) were examined in the present study using the sand maze. It was hypothesized that any deficit produced by WIN-2 during training would be diminished by returning the subject to the original learning state during testing (i.e., no drug). Figure 1 Figure 2 Discussion All rats performed equally well during shaping. Rats in the same state groups preferred the correct quadrant more than rats in the different state group. Rats in the different state group appeared to exhibit a place aversion for the correct quadrant. Robinson, et al., 2003 Future research might examine the state dependent properties of WIN-2 with a lower dose of the drug (e.g., 3 mg/kg) to reduce the possible aversiveness produced by larger doses. Method Subjects The subjects were 90-day old, male Long-Evans rats (N=20). Rats were reduced to and maintained at 85% of their free-feeding weights one week prior to and during the experiment. Water was available ad libitum. Apparatus The sand maze was a plastic pool (36 inches wide by 6 inches deep) filled with a sand/crushed Froot Loops (FL) cereal mixture that was 2 inches deep (approximately 11 ounces of FL was crushed and mixed with 100 pounds of play sand to make the mixture). The maze was elevated 36 inches off the floor. Procedure Each rat was placed in the sand maze and required to find FL, which were located in one consistent location during shaping and training (i.e., NW, NE, SW, or SE quadrant). Rats were started from a different location in the maze on each trial (i.e., N, S, E, or W). After finding the reward on any given trial, the rat was allowed to consume approximately three to four FL prior to termination of the trial. Rats were handled prior to shaping. A three-day procedure was used with two trials per day, which included shaping, training, and testing (see Table 1). Rats were randomly assigned to groups that determined whether they were injected with WIN-2 (5.6 mg/kg) or VEH during training and testing (see Table 1). Rats received intraperitoneal (I.P.) injections of WIN-2 (5.6 mg/kg) or VEH 30 minutes prior to training and testing trials. Latency to find the reward was measured during acquisition. Latency to dig and quadrant preference were measured during videotaped test trials. References Hanson, G.R. (2003).  The sand maze: An appetitive alternative to the Morris water maze (Doctoral dissertation, Kent State University, 2003). Dissertation Abstracts International, 63, 4958.   Lichtman, A. H., Dimen, K. R., & Martin, B. R. (1995). Systemic or intrahippocampal cannabinoid administration impairs spatial memory in rats. Psychopharmacology,119(3),282-290. Morris, R.G.M. (1981). Spatial localization does not require the presence of local cues. Learning and Motivation, 12, 239-260. Robinson, L., Hinder, L., Pertwee, R. G., & Riedel, G. (2003). Effects of Delta-9- tetrahydrocannabinol and WIN-55,212-2 on place preference in the water maze in rats. Psychopharmacology, 166, 40-50. Spear, N. E. & Riccio, D. C. (1994). Memory: Phenomena and Principles. Boston: Allyn & Bacon. Figure 3 Table 1 Day 1: Shaping Trial 1: Exposed Trial 2: Partially Buried Day 2: Training Trial 3: Shallow Buried Trial 4: Buried (Shallow) Day 3: Training Trial 5: Buried (Medium) Trial 6: Buried (Deep)  Day 10: Testing Probe Trial (No Reward Buried in the Sand) Different State (n=6) No drug WIN-2 Same State (n=5) VEH (n=9)