We compared the Plus and Zero mazes in adult Sprague-Dawley rats to determine if there are differences in their detection of behaviors associated with approach-avoidance conflict and as a function of treatments documented to change such behavior in opposite directions. Both mazes were constructed according to published dimensions (Pellow et al. 1985
;Shepherd et al. 1994
). Open area curbs were not present in the original Pellow et al. Plus design but were subsequently shown to contribute to open area exploration (Fernandes and File 1996
); curb height was standardized in our mazes. While the elevation of our mazes differed, this has been shown to have no significant effect on open time (Treit et al. 1993
). Both the Plus and Zero were equally effective at reflecting the anxiogenic effects of restraint, yohimbine, and caffeine and the anxiolytic effects of diazepam. The only discrepant finding between mazes following challenge was that diazepam-treated groups in the Plus showed a decrease in closed area entries not seen in the Zero. However, all indices related to anxiety were identical between mazes and no detection of effect differences were observed.
Several treatments (pharmacological and behavioral) were included to increase the generality of the findings (Pellow et al. 1985
). In line with approach-avoidance concepts, the percentage of time spent in the open areas of both mazes was independent of the level of overall locomotor movement, and rats spent more time in closed than open areas, consistent with the idea that open areas evoke anxiety resulting in avoidance behavior (Lister 1987
;Pellow et al. 1985
;Rodgers et al. 1997
;Rodgers and Dalvi 1997
;Shepherd et al. 1994
). In this experiment, each treatment, except for nicotine, altered percentage time spent in open areas in the expected direction in both mazes; that is, decreasing it after yohimbine, caffeine, or restraint exposure and increasing it after diazepam treatment. These data support the use of percent time in open areas (or open time in the Zero) as the principal index anxiety-related behavior. The number of head dips has also been suggested to be an index of anxiety in rats (Cole and Rodgers 1995
;Cruz et al. 1994
;Fernandes and File 1996
;Rodgers and Johnson 1995
), and in agreement with this, restraint, yohimbine, and caffeine all produced significant reductions in head dips.
Nicotine, at the dosage used here, had no effect. An identical dose of nicotine has previously been shown to increase anxiety in the Plus, although there were several differences between that study and ours that could have contributed to this, including handling prior to maze testing in the previous study, lighting differences within the testing room, materials used to construct the mazes, and maze cleaning procedures (Hogg 1996
;Irvine et al. 2001
). Difference in rat strain and age between studies could also have been contributing factors (Hogg 1996
;Imhof et al. 1993
). All of these were controlled within the present experiment so that comparisons across mazes could be made, and no main effect of maze was shown following the treatments used here.
In addition, both male and female untreated rats performed similarly in the two mazes. Lister female rats (60–70 days old) have previously been shown to spend increased percent time in open areas of the Plus (Johnston and File 1991
). While it has been suggested that, in the Plus, females are more active and exploratory as opposed to being less anxious (Fernandes et al. 1999
), other studies have not shown significant differences in male and female performance in this test in naïve animals or following prior testing (Doremus-Fitzwater et al. 2009a
;Doremus-Fitzwater et al. 2009b
;Wilson et al. 2004
). This difference between studies on the effect of sex could be due to strain or age differences as well as estrous cycle in females (Imhof et al. 1993
;Reddy and Kulkarni 1999
;Rodgers and Cole 1993
;Trullas and Skolnick 1993
). Care should be taken with regard to these factors in future studies. The use of females here was not to determine so much whether sex was a significant variable within mazes, but whether there were differential patterns between mazes. The data showed no such differential effect.
In untreated rats, the Zero produced higher exploration in open areas than the Plus, suggesting that some factor in the Plus may inhibit exploration of open areas more than in the Zero, perhaps because of the time spent in the central region in the Plus. Examination of , suggests that latency to first open entry was also increased in the Plus and this may explain some of the differences in open area exploration observed between mazes. The difference in these baseline patterns may make the Zero more sensitive to some treatments than the Plus but this will require further investigation to prove. We also note that SAL-treated animals across experiments showed differences in time in open in the range of 18–45%. By contrast, the SAL-treated animals in the Plus showed time in open in the range of 16–25% which was more consistent across experiments. However, when testing drug effects, the data showed that the mazes were comparable at detecting differences.
The relative sensitivity of these two mazes to detect alterations in anxiety through dose-response experiments was not included here. While our experiment was in progress, we identified two studies that compared the Plus and Zero in mice using anxiolytic and anxiogenic drugs at multiple doses (Kulkarni et al. 2007
;Kulkarni et al. 2008
). While Kulkarni et al. found the Zero to be more sensitive to benzodiazepines (i.e., the Plus showed significant drug effects at more dose levels of each drug), they did not report the time spent in open areas of the Plus as a percentage, nor did they include the amount of time mice spent in the center region, making cross-maze comparisons difficult. However, mice tested in either the Plus or Zero were shown to have altered anxiety profiles following pharmacological manipulation, implying that the testing of additional doses in rats is unlikely to change our conclusions.
To account for possible locomotor activity differences in addition to measuring closed area entries, locomotor activity was assessed in locomotor chambers for 30 min immediately following maze testing. With the exception of nicotine, the time span for locomotor testing was within the half-life of each drug, whereas for nicotine testing the first 5 min was within its half-life (Banna et al. 2010
;Ghosheh et al. 1999
;Lau et al. 1995
;Loscher and Schwark 1985
). Nicotine treatment did not alter the number of closed area entries in either maze or distance traveled in the locomotor chambers. Restrained animals as well as caffeine- and yohimbine-treated animals had no overall difference from controls in locomotor activity, but in both mazes had decreased entries into the closed areas, suggesting some reduction in activity at short time intervals after drug administration that appeared during maze testing but was absent during locomotor activity testing. Consistent with diazepam-induced hypoactivity previously reported (Nishino et al. 2008
), we found diazepam-induced decreases in closed area entries in the Plus, but no changes in the Zero. Only diazepam-treated animals showed a decrease in distance traveled in the activity chambers compared to SAL-treated controls, but ANCOVA showed this hypoactivity did not correlate with anxiety measures. The ANCOVA was performed for each treatment using either the first 5 min or the entire 30 min of locomotor testing as the covariate. The first 5 min interval of locomotor testing is the most immediate to maze testing and the most likely to reflect general drug effects although the half-life for each drug, with the exception of nicotine, fell within the 65 min total testing time (30 min from injection to maze start, 5 min Plus or Zero maze testing, 30 min activity testing). Neither ANCOVA on the first 5 min nor 30 min altered the difference in percentage of time spent in the open for any group supporting the efficacy of both mazes in separating anxiety from activity. Without the locomotor test, the anxiogenic effect of caffeine might have been attributed to an activity difference because of the decreased number of closed area entries. This could support the use of an independent measure of activity in addition to determining the number of closed area entries for compounds that are likely to induce some level of locomotor change. An alternative approach to using a separate index of activity is counting the number of line crossings, photocell interruptions, or path length using tracking devices made by animals within both regions of the mazes. Others have used these methods in lieu of closed area entries thereby removing anxiety measures from activity scores (Jacobson and Cryan 2008
) and it appears that simultaneous measurements of this kind are preferable. This was not possible in this study due to the camera angle of our video recording system using a one camera arrangement. Therefore, utilizing locomotor activity in situations where within zone activity measurements are not possible may provide some useful information.
We also analyzed the first 5 min of locomotor activity testing for time spent in the periphery versus center region, which is often used as an index of anxiety. Only caffeine exhibited an altered activity in the center region, however peripheral activity was also decreased, hence, the effect was non-specific. These results were consistent with the overall locomotor findings as opposed to the maze findings; indicating that central zone activity in the apparatus we used may not be as sensitive as the Plus and Zero mazes for assessing anxiety when given after maze testing. Being handled and tested in either the Plus or Zero prior to open field testing could potentially have altered performance on the latter test. Since maze and locomotor testing order was not counterbalanced exact determination of order effects cannot be obtained from these data.
The main difference in the Zero is that it removes the center region found in the Plus. Depending on treatment, animals tested here in the Plus spent 13% to 30% of their test time in the center, thereby decreasing the time spent in open or closed areas by 39–90 s of the total 300 s test session. Differing levels of anxiety could present themselves based not on the amount of time spent in an open area, but in the amount of time spent in the center, thereby increasing the percentage of time spent in the open areas. The Zero allows time spent in the open areas to be expressed in direct proportion to total test time without the need to convert it to percentage open time. Since time spent in open is the measure of principal interest, it is important that Plus data be expressed this way: unfortunately, this is not always the case (Drapier et al. 2007
;Frye et al. 2008
;Koks et al. 2001
;Kompagne et al. 2008
;Kulkarni et al. 2008
;McDermott and Kelly 2008
;Nosek et al. 2008
). This more direct measure of time in open, therefore, represents an advantage of the Zero maze.
This is the first experiment to compare the elevated Plus and Zero mazes in rats. The main advantage of the Plus is the historical data available. The present results indicate that when observing pharmacological or behavioral manipulations on anxiety, if time spent in the center region in the Plus is excluded such that time in the open is represented as a percentage, the results from both mazes are essentially equal for the independent variables evaluated here. The slight advantage in the Zero with higher percent time in the open may be important in some contexts since in many experiments detection sensitivity is a function of the baseline level of performance relative to control groups. Since the Zero often showed a higher baseline, detecting change in both directions may be easier in the Zero such as when comparing basal levels in transgenic models.