Randolph-Macon Woman’s College 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 Figure 1 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). Results Acquisition All rats met the acquisition criterion. As seen in Figure 2, there were no significant differences between groups in latency to find FLs during any trials (Trial 1: F(3,17)=.826, p>.05; Trial 2: F(3,17)=1.440, p>.05; Trial 3: F(3,17)=1.201, p>.05; Trial 4: F(3,17)=1.017, p>.05; Trial 5: F(3,17)=1.436, p>.05; Trial 6: F(3,17)=1.031, p>.05). Testing As shown in Figure 3, there were no significant differences between groups in total quadrant preference on the probe trial test (F(3,17)=.367, p>.05). In addition, none of the groups differed from chance on the test trial (WIN/WIN: t(6)=-1.360, p>.05; WIN/VEH: t(6)=.428, p>.05; VEH/WIN: t(2)=-.240, p>.05; VEH/VEH: t(3)=-.594, p>.05). Day 1: Shaping Trial 1: Exposed Trial 2: Partially Exposed Day 2: Training Trial 3: Partially Buried Trial 4: Shallow Buried Table 1 Day 3: Training Training Trial 5: Buried Trial 6: Deep Buried WIN/WIN (n=7) SDR VEH/WIN (n=3) Control WIN/VEH Amnesia VEH/VEH (n=4) Testing Day 10: Testing Probe Trial: No FL

Method Discussion Figure 2 Subjects The subjects were 90-day old, male Long-Evans rats (N=21). Rats were reduced to and maintained at 85% of their free-feeding weights one week prior to and during the experiment. Rats were maintained on a 12-hour light/dark cycle. 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 Froot Loops cereal (FL), which was 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 for one week prior to shaping. A three-day procedure was used with two trials per day, which including shaping, training, and testing (see Figure 1). Prior to shaping, rats were randomly assigned to groups which determined whether they received an injection of WIN-2 (3.0 mg/kg) or VEH during training and testing (See Table 1). Rats received intraperitoneal (I.P.) injections of WIN-2 (3.0 mg/kg) or VEH 30 minutes prior to training and testing. Latency to find the FL was measured during acquisition, rats were required to find the FLs within 10 minutes of the start of the trial in order to meet the acquisition criterion. Once acquisition criterion was met, rats received a test trial in which no FL were buried in the maze. Latency to dig and quadrant preference were measured during videotaped test trials. Discussion All rats performed to the same degree during shaping and training. None of the groups exhibited memory for the task during testing, regardless of drug administration. Lack of memory may be due to the training procedure employed, specifically massed training trials Trial Spacing Effect: Kraemer & Randall (1985) Figure 3 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.   Kraemer, P. J., & Randall, C. K. (1985). Spatial learning in preweanling rats trained in a Morris water maze. Psychobiology, 23(2), 144-152. 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. Spear, N. E. & Riccio, D. C. (1994). Memory: Phenomena and Principles. Boston: Allyn & Bacon.