After this, the demon would soon set its plan into motion. Those in the town of Haven who fell asleep ran the risk of never waking. This "sleeping sickness" soon became an epidemic within the town and baffled all experts who were unaware of its real cause but advised citizens to remain calm. Over twenty years went by and more people continued to fall asleep. Some tried to resist sleeping but eventually gave in due to exhaustion. Those who were asleep became trapped in their dreams and subjected to the demon's will, terrorized by horrific nightmares. These nightmares were part of the demons plan to select special people who would willingly submit and become its chosen sleepers. Several of these people would perish in their dreams and subsequently in the real world as well. Those that survived and submitted, however, were allowed to live in a dreamworld that put on the facade of being a good dream or that they were awake but in actuality, was a place under the demon's control and where it would keep the chosen sleepers for its plan. The demons endgame in all this was to amass three hundred and sixty chosen sleepers in order to bring itself to life. Using the sleepers, the demon was also able to create a dream barrier around the whole town of Haven which prevented anyone from leaving. The people of Haven, both awake and sleep, remained the demon's prisoners as it came closer and closer to its goal. Meanwhile, Tom secretly continued to serve the demon as an adult, albeit reluctantly. Tom obeyed the demons orders and kept watch over the sleeping townsfolk while keeping the few still awake in check.
I heard I could get out of a maze using just one side of hands, just following the one side of a wall of a maze. It probably is from math, then what kind of subject is related to a maze? Perhaps Topology? I'm trying to think whether this kind of solution is related to a system of an creature or an individual.
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An open field maze consists of a wall-enclosed area that is of sufficient height to prevent the subject from escaping. Typical maze shapes are circular or square with an area large enough, based on the size of the subject tested, to elicit a feeling of openness in the center of the maze. A number of variables can be scored in the open field maze with most parameters involving differing types of motor activity2. Ambulation is the most common behavior studied but others such as latency or rearing can also be measured. Most often, rodent behavior is analyzed in a bare maze. However, the addition of objects, either one or many to the maze floor, adds the ability to see how the subject interacts with novel additional stimuli2. Relevant parameters when objects are presented are typically the number of approaches to an object or in some cases, preference or aversion for one object over another.
The first and arguably most important specific parameter to measure in the Open Field Maze is total ambulatory distance. While the unit of measure is irrelevant for comparison purposes, it is most often expressed as a metric measurement (cm). In the experimental data presented here (Figure 5), Wild type (WT) or normal C57BL/6 mice show similar ambulatory ability to a specific knock-out C57BL/6 mouse strain (KO). When total distance traversed is similar between strains or treatments, further analysis of emotional behaviors is simplified because locomotor activity is effectively removed from the equation. If there are significant differences in the locomotor ability of the mice tested, further analyses such as zone entries or time spent in certain designated zones of the maze can be skewed due to inactivity instead of strain or treatment effects. Techniques exist to account for unequal locomotor activity but these are most often specific to the research question.
As total ambulatory distance between the two mouse strains was similar, we were able to analyze thigmotaxis, or the tendency of a subject to remain close to walls, in the WT versus KO mice (Figure 6). The degree of thigmotaxis has been validated as a measure of anxiogenic behavior in mice10. Thigmotaxis increases as anxiety levels rise. Using the SMART software, individual zones were overlaid on the paths traveled by the mice (Figure 3) and time spent in inner zones versus outer zones calculated and presented as a function of total time (10 min) in the maze. In this case, KO mice displayed significantly higher anxiogenic behavior than their WT counterparts. A representative travel path can be seen in Figure 4 where the WT mouse path traverses the middle areas of the maze at a much greater frequency than does the KO mouse which remains close to the walls of the maze even though the distance traveled of both mice is similar. We can conclude from this that the KO mice exhibit higher anxiety associated behavior than do WT mice.
To further support increased anxiety levels in the KO mice, fecal boli left in the maze after the 10 min test period were counted by the observer once the test subject was removed (Figure 13). Following the popular view that highly emotional animals exhibit increased defecation, KO mice exhibit significant increases in fecal boli present when compared to WT mice. This correlates with the levels of thigmotaxis measured in the KO mice and indicates that the knock-out mice used in this study showed increased emotionality and anxiety when compared to their WT counterparts.
Figure 1.Example of a four quadrant OFM. The OFM pictured was obtained from San Diego Instruments and was used in all testing procedures described. Dimensions of one quadrant of the maze are in centimeters and each quadrant is identified (Q1-Q4).
Figure 3. Comparison of track paths for WT and KO mice. Representative tracks for either WT or KO mice are shown. Each track represents the total distance traveled by the subject during the 10 min time period of the test. The beginning point (B) and the end point (E) of the tracking is indicated. The WT example track crosses into the center portion of the maze at regular intervals while the KO track remains closely in proximity to the walls of the maze indicated increased thigmotaxis or anxiety-related behavior.
Figure 4. Zone overlay used to interpret tracking data for thigmotaxis from the OFM. Using the SMART software analysis package, a series of 10 x 10 cm zones were identified and used to evaluate subject tracks. The outer zone consisted of 16 blocks as identified while the inner zone consisted of 9 blocks and is shaded. Greater time spent in the outer zones of the maze is recorded as increased thigmotaxis and is indicative of amplified anxiety-related behavior.
Figure 5. Total distance traveled in the OFM. WT (n = 24) and KO (n = 27) mice were subjected to the OFM and total distance in centimeters of their respective tracks were combined and statistically analyzed to visualize any differences in ambulation. WT and KO mice performed similarly in the OFM when total distance was measured. The results for the data were expressed as the mean +/- S.E.M. Statistical analyses (t-tests) were performed using Excel 2010 (Microsoft, Redmond WA) and SAS 9.2 (SAS Institute, Cary NC). [Data is modified from Ramesh Babu, et.al., 2008.]
Figure 6. Time spent in inner and outer zones of the OFM. WT (n = 24) and KO (n = 27) mice were subjected to the OFM and time spent in inner and outer zones of the maze statistically analyzed for differences in mouse strains. Time spent in the outer zones of the maze identified in Figure 7 measures thigmotaxis or wall-hugging behavior and is indicative of anxiety-related behavior. KO mice exhibited higher anxiety measures than to WT based on thigmotaxis. The results for the data were expressed as the mean +/- S.E.M. Statistical analyses (t-tests) were performed using Excel 2010 (Microsoft, Redmond WA) and SAS 9.2 (SAS Institute, Cary NC). p
Figure 7. Fecal boli deposits in the OFM. WT (n = 24) and KO (n = 27) mice were allowed to complete a 10 min test in the OFM before being removed from the maze back to their home cages. After mice were removed, the number of defecations or fecal boli deposits was manually counted by the observer. Increased number of boli can be indicative of increased anxiety and emotionality of the subject animal. KO mice exhibited an increase in fecal boli when compared to WT. The results for the data were expressed as the mean +/- S.E.M. Statistical analyses (t-tests) were performed using Excel 2010 (Microsoft, Redmond WA) and SAS 9.2 (SAS Institute, Cary NC).p
Here, three aspects of open field behavior are readily characterized using this protocol: 1) Total distance covered (in cm) during the entire timed portion of the test; 2) Thigmotaxis or a measure of the percent of the 10 min total test time that the subject remains adjacent to the outer wall of the maze which is indicative of anxiety-like behavior; 3) The number of fecal pellets (boli) left in the quadrant after the subject is removed is counted. Defecation is a negatively related measure of emotionality in rodents5 and can be used to indicate levels of anxiety in the mouse subject. A brief instruction on how to access these measurements in the SMART software follows.
Locomotor activity of the test subjects is important to discern prior to analysis of OFM data or for that matter, any animal behavioral maze. When comparing different strains of mice or different effects of drug treatments, the ambulatory ability of the mouse is paramount. If locomotor ability is compromised due to treatment effects, then measuring activities that rely on the ability of the subject to move is confounded. Therefore the first step in this experiment was to compare total movement between the two subject strains. Using the subject tracking feature of the SMART software, we measured the total ambulatory distance covered in the maze during the duration of the test by two different strains of C57BL/6 mice (Figure 4). Both Wild Type (WT) and genetic knock-out mice (KO) displayed similar ambulatory ability. There was no statistical difference in total distance (in cm) traveled by either mouse line throughout the 10 min time frame of the experiment. Had one strain shown a significant difference in ambulation compared to the other, a more specialized investigation, possibly using other behavioral paradigms, would be required to characterize the difference. However, in this case, ambulation of the subject strains was equal allowing direct use of unadjusted OFM-derived data to further investigate anxiolytic parameters. It should be noted here that some researchers have interpreted high activity or increase exploratory behavior as an index of low emotionality while others conceive of exploratory behavior being independent of emotionality11. One has to acknowledge that differences in locomotor activity can confound emotional measures12. However, as total ambulatory distance was similar between the mouse strains used here, activity levels of the mice were separated from emotionality factors. 17dc91bb1f
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