Simple Protocol for Distinguishing Drug-induced Effects on Spatial Memory Acquisition, Consolidation and Retrieval in Mice Using the Morris Water Maze

Abstract

The Morris water maze (MWM) is one of the most commonly used tests for assessing spatial learning and memory in mice. While the MWM is highly amenable to testing the effects of memory modifying drugs, most studies do not consider the timing or duration of drug exposure when conducting the MWM assay; factors that can strongly influence the effect of the drug on different stages of memory and interfere with data interpretation. Herein we describe a MWM protocol which offers the advantage of distinguishing the impact of a fast acting intraperitoneally (IP) injected drug on the different stages of spatial memory: acquisition, consolidation, and retrieval. Mice initially undergo habituation to both the MWM apparatus and IP injection procedure over the course of three days. For assessing the effect of a drug on memory acquisition, mice are injected with the drug prior to training sessions over four consecutive days, where mice learn to find an escape platform in a circular water tank using distal spatial cues. To determine the effect of the drug on memory consolidation, mice are injected with the drug immediately after each training session. For testing the effect of a drug on memory retrieval, mice receive mock IP injections on each training day and the drug is IP injected only once, prior to a probe trial, where mice attempt to locate the platform following its removal from the tank. This protocol provides a simple strategy for distinguishing the effect(s) of a CNS acting drug on the different stages of memory.

Keywords: ANY-Maze; Drug intervention; Fast acting drug; Intraperitoneal injection; Learning; Morris water maze; Simplified method; Spatial memory.

Figure 1.. Holding and testing area setup.

The tank must be curtained/walled off such that the holding area and experimenter are not visible to mice in the tank. Distinct spatial cues in the testing areas must be displayed at a height visible to the mice from within the tank. The digital camera is mounted above the center of the tank (not depicted).

Figure 2.. Platform and mouse placement.

The starting location of mice beginning each trial is indicated by a hand holding a mouse. During all trials starting on day 3 and onward, mice are to begin facing the wall of the tank. The initial location chosen to start mice on day 3 determines the relative location mice are to begin each trial on subsequent days. The small grey circle indicates the location the submerged platform within the tank.

Figure 3.. Behavioral testing timeline.

Injection regime for testing the effect of drug on either memory acquisition, consolidation or retrieval separately. Arrows of indicated colors specify when drug injections are to occur. When testing drug impact on acquisition or consolidation, mock injections with a ballpoint pen poke mimicking an intraperitoneal injection occur prior to probe 1, probe 2 and the flag trials. When testing drug impact on retrieval, mock injections occur prior to each day of training. For all testing conditions, mock injections occur during habituation.

Figure 4.. Representative Data for drug-mediated effects on memory acquisition.

Average latency to find the platform (A), path length (B) and mean speed (C) for mice across all four trials for each training day. Total distance traveled (D) and percent time spent in target quadrant (E) for mice on Probe 1. The number of times mice enter the area where the platform was previously located during training for probe 1 (F) and probe 2 (G). Latency for mice to find the flagged platform on the final flag trial (H). Data is presented as mean and SEM (n = 7). No less than seven mice are recommended for each group. However, a larger number of mice may be required in order to detect statistically significant differences between drug and vehicle treated mice if the effect is small. Example data is provided demonstrating a drug showing impact on memory acquisition. Graphs of the same format are to be produced separately for tests of drug impact on memory consolidation and retrieval as well. Injection with drugs showing an impact on either memory consolidation or retrieval is not expected to result in significant differences in latency to find the platform and path length, as seen in (A) and (B) respectively, but is expected to result in reductions of percent time in target quadrant, as seen in (E), or the number of platform entries, as seen in (F) and (G) during probe trials.