The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.
The elevated plus-maze was derived from the early work of Montgomery.27 He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley and Mithani, and File et al.22,29 performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice25. It is now a standard assay for anxiolytic treatments23, and several protocols have been published36,24 http://www.jove.com/video/1088; http://currentprotocols.com/protocol/ph0538.
The elevated plus-maze consists of two opposite open and two opposite closed arms intersecting at a central area, in the form of a + in plan. Percentage time spent on, and percentage entries into, the open arms are an index of anxiety-like behaviour; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious.
Factor analysis has shown that while indices of open arm activity load heavily on a factor designated as anxiety-like behaviour, and closed arm entries largely reflect motor activity, the boundaries between factors are not definitive; also other factors such as exploration, risk assessment and decision making comprise the profile of behaviour on the plus-maze,12,32. Time in the central area is more problematic to interpret, although time spent here has been classified as “decision making”32. Although mice spend most of their time in the enclosed arms, and relatively little time on the open arms, in many studies much time is spent in the central area where the arms join. Although this may represent decision-making32, it is ambiguous in terms of anxiety-like behaviour, although highly anxious mice might be expected to avoid the central area as well as the open arms. An early attempt to eliminate this ambiguous central area was to construct a circular maze with two closed quadrants alternating with two open ones33. In this “zero-maze” an animal is unambiguously in an open (anxiogenic) or a closed (nonanxiogenic) zone. However, although used by several groups, it has never been as widely adopted as the plus-maze. In our laboratory we found that whereas the plus-maze (and successive alleys) showed anxiolytic effects in hippocampal lesioned rats, these were not seen on a zero maze constructed exactly like that of Shepherd et al.13
The successive alleys apparatus was therefore devised with the primary aim of eliminating the central area of the plus-maze. Secondarily, the elements of other tests of anxiety-like behaviour were incorporated. By starting the animal in the enclosed alley 1, an emergence component was introduced, similar to that in Stone’s “stovepipe” test34. The color of the alleys changes from black (alley 1) through grey (alley 2) to white (alleys 3 and 4). This originates from observations that mice prefer darker areas, as exemplified in the light-dark box test11. The walls of the successive alleys also slowly decline in height, unlike in the plus-maze where there is an abrupt transition between high walls and no walls. The width of the alleys progressively decreases to gradually increase the exposure of the mouse to the elevated position of the maze, in concert with the gradual decrease in wall height. All these factors were incorporated in an attempt to increase the sensitivity of the test and span a wider range of anxiety-like behaviour than the plus-maze does. The successive alleys apparatus was an attempt to bracket a wider range of anxiety-like behaviour baselines, in the same way that the four baseline conditions of the social interaction test as originally performed by File17 permit detection of subtle modifications of anxiety-like behaviour. File, in a study of the effects of nicotine, only saw differences in the two intermediate anxiety-like behaviour conditions, not the highest or lowest20.
As well as the plus-maze, another widely used test of anxiety-like behaviour is the light-dark box11. Results from the light-dark box and the plus-maze do not always correlate with each other35. It is now considered that anxiety-like behaviour is not a unitary phenomenon19. The successive alleys test may therefore well give results different to those obtained using the light-dark box, and conceivably even from the plus-maze, since it is subtly different from the latter; however, results between the former and the latter would be expected to be broadly similar.
It has been shown that repeated testing on the plus-maze alters the type of anxiety observed.8,21 It may be that exposing an animal to the plus-maze followed by the successive alleys eliminates the change in the type of anxiety-like behaviour measured if the plus-maze test is repeated. An example of this was seen when the plus-maze and successive alleys were used to test medial prefrontal cortex-lesioned and control mice in a temporally counterbalanced manner. The lesioned mice showed significantly lower anxiety-like behaviour in both types of apparatus, regardless of whether testing occurred with the successive alleys or the plus-maze first15. It is also possible that sequential use of the plus-maze and successive alleys might preserve the sensitivity to anxiolytic drugs seen when the plus-maze test is repeated.18
Rats can also be tested on a larger version of the successive alleys. The procedure is exactly the same as for mice.
As in the case of testing for hyponeophagia, the prior general experience of the animals, apart from elevated maze anxiety-like behaviour testing per se, may affect the result.1,16
In conclusion, the successive alleys test produces generally similar results to the plus-maze when lesions of the hippocampal complex or medial prefrontal cortex are made, but further work is needed to assess the effects of lesions in other brain areas thought to be related to anxiety-like behaviour. Occasionally, results on the plus-maze differ markedly from those in the successive alleys; an example of this is our work on Kir6.2 KO mice14. When they were tested on both apparatuses, it allowed us to demonstrate that KO mice did not show a general unidirectional change in anxiety-like behaviour, since they spent more time than wild-type mice on the open plus-maze arms (showing less anxiety-like behaviour), but more time in alley 1 of the successive alleys (showing greater anxiety-like behaviour). Thus, combined use of the plus-maze and successive alleys can eliminate false positive or false negative results, since the two tests, although similar, are not functionally identical.
As in the plus-maze, hyperactivity does not lead to a non-selective increase in the number of alleys entered. Studies have shown that the organization of behavior in mice varies independently of activity 28. Medial septal lesioned rats, which were shown to be markedly hyperactive in a separate test in photobeam activity monitoring cages only showed significantly greater entries into alleys 2 and 3; entries into alleys 1 and 4 were not different to controls.5 So the less anxious septal lesioned rats were shuttling back and forth between the two moderately anxiogenic areas of the successive alleys, showing what the apparatus was designed to detect, i.e. their position on a spectrum of anxiety. If they had been even less anxious, an increase in crossings between alleys 3 and 4 would have been seen.
Other evidence that hyperactivity does not lead to a false positive in the successive alleys test is that rats with dorsal hippocampal lesions are hyperactive but do not show anxiolytic effects in the successive alleys7. In a related test with linear apparatus, the black-white alley test, GluRA KO female mice show rather more anxiety-like behaviour than controls while males are similar to controls, yet both sexes are markedly hyperactive3. Kir6.2 KO mice were more active in their home cages than wild type mice but showed les anxiety in the successive alleys14. Notably, 8 KO mice jumped or fell off the plus-maze but none from the successive alleys.
One limitation of the successive alleys test as presently used is that, given the varying color of the floor, video-tracking systems would be difficult to use. However, the floor could probably be painted a uniform color as color is only one of the factors in the apparatus that generates anxiety.
The successive alleys test is unlikely, however, not to suffer from the variability problems associated with similar ethologically based tests such as the plus-maze10,23, but testing a batch of animals on both tasks might help to clarify an inconclusive plus-maze test30,31,37; a multiple-test approach may have advantages when testing anxiety-like behaviour.
The authors have nothing to disclose.
The Wellcome Trust for providing Open Access funding to Oxford University. Robert Deacon is a member of Oxford OXION group, funded by Wellcome Trust grant WT084655MA.