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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: J Neurosci Methods. 2021 Aug 18;363:109325. doi: 10.1016/j.jneumeth.2021.109325

Light-Dark Open Field (LDOF): A novel task for sensitive assessment of anxiety

Khadijah Shanazz a,b, Rachael Dixon-Melvin a,b, Kristopher M Bunting a,b, Rebecca Nalloor a,b, Almira I Vazdarjanova a,b,*
PMCID: PMC8878097  NIHMSID: NIHMS1779100  PMID: 34418444

Abstract

Background:

Pre-clinical studies of psychiatric disorders often include a measure of anxiety-like behavior. Several tasks exist that serve this purpose, but because anxiety is complex with a myriad of anxiogenie stimuli, researchers are often compelled to use multiple tasks. The Light-Dark Open Field (LDOF) combines concepts from two such tasks, Light-Dark Box and Open Field, into one task with the synergistic effect of enhanced discrimination of anxiety-like behavior.

New methods:

Our goal was to increase the sensitivity of the Open Field task with the addition of a shadow, conceptually similar to the Light-Dark Box, to detect concealed differences even under bright light, which is highly anxiogenie. The resulting LDOF allows assessment of anxiety due to bright light and open space simultaneously, while retaining the ability to assess the impact of each with custom indices. In addition, it maintains all the advantages and measures of the Open Field.

Results:

Using custom created indices from measures collected in the LDOF one can assess anxiety induced by light, open space, or light and open space combined and thus elucidate anxiety-inducing factors. Using two strains of rats: an outbred strain, Sprague-Dawley (SD), and a strain that exhibits high trait anxiety, Lewis rats, we found that increased discrimination for anxiety-like behavior can be achieved with the Light-Dark Open Field.

Comparison with existing models:

The LDOF allows researchers to extract the traditional measures of an Open Field, including valuable information about locomotion and habituation while adding a further layer of discrimination with the light-dark component. Because the LDOF is a combination of two different tests, it saves time compared to running multiple experiments in series that then need to be counterbalanced to reduce artefacts and task order effects. In addition, it detects differences even when traditional tasks of anxiety have reached their ceiling sensitivity (i.e. EPM under bright light conditions).

Conclusion:

We present the Light-Dark Open Field: a simple modification of an existing Open Field apparatus that incorporates aspects of the Light-Dark Box with the addition of a shadow. The shadow (Dark Perimeter) allows for increased discrimination in detecting anxiety-like behaviors. Comparison of anxiety-like behavior between Lewis and SD rats allowed us to develop the construct and face validity of the LDOF as well as demonstrate its sensitivity even under bright light conditions. In addition, we developed 3 indices that allow one to parse out, from one set of data, the effect of two anxiogenie stimuli- bright light and open space.

Keywords: Light-Dark Open Field, Open Field, Elevated Plus Maze, Anxiety, Behavior, Light Anxiety, Open Space Anxiety, Rats, Sprague Dawley rats, Lewis rats

1. Introduction

Anxiety is a state of fear and worry associated with the anticipation of a future threat (American Psychiatric Association, 2013) and is a component of many psychiatric disorders including anxiety disorders such as panic disorder, separation anxiety disorder, social anxiety disorder, generalized anxiety disorder; post-traumatic stress disorder, depression, and obsessive compulsive disorder (Craske et al., 2017). Anxiety is often considered as a combination of trait anxiety, an innate characteristic of the individual, and state anxiety, anxiety experienced at a particular moment in the presence of an anxiogenie stimulus (Saviola et al., 2020; Steimer, 2011). Anxiety disorders alone affect nearly 34% of the population (Bandelow and Michaelis, 2015) and cause extreme emotional distress. Alarmingly, the prevalence is higher in the younger generations, including college students (Lipson et al., 2019). Importantly, anxiety is not a unitary concept and can be induced by different factors with varying symptomology and severity (Bandelow et al., 2017). It is therefore important to understand both the pathophysiology of anxiety and parse out contributing sources of anxiety as well as test potential new treatments with preclinical investigations.

In animal models, anxiety-like behavior is measured with the use of behavioral tasks. Measures associated with anxiety in animals are necessarily observations of changes in behavior in the presence of anxiogenic stimuli (Beuzen and Belzung, 1995; Lister, 1990; Steimer, 2011). Because rodents are ethologically nocturnal animals that tend to show a preference for dark enclosed spaces, assessing anxiety in rodents involves measures of unconditioned approach, avoidance, and “risk assessment” behaviors such as their willingness to venture into open, well-lit spaces, and/or high places (Crawley, 1981; Pi et al., 2020).

To accommodate the complex nature of measuring anxiety-like behavior, researchers have developed several different tasks for measuring this behavior in rodents that utilize different anxiogenic stimuli (Ramos, 2008). For instance, the Light-Dark Box, a box divided into two sections, one well-lit and the other dark, uses measures of time spent in the light or latency to enter the light to infer anxiety-like behavior (Bourin and Hascoët, 2003; Crawley and Goodwin, 1980; Kulesskaya and Voikar, 2014). The Elevated Plus Maze (EPM), a commonly used task for measuring anxiety-like behavior, consists of two enclosed arms and two open arms with two common configurations; a plus sign “+” / “x” shape or a circle, elevated from the floor. Time spent in the open arms, latency to enter the open arms, and number of entries into the open arms are measures of anxiety-like behavior used in the EPM (Kraeuter et al., 2019; Pellow et al., 1985; Pellow and File, 1986). Another test, Social Interaction, is also known to be affected by anxiety state such that a more anxious rat will have fewer social interactions than their less anxious counterparts (Bailey and Crawley, 2009; File and Hyde, 1978). The Open Field test uses an open arena where the animal is allowed to freely explore for some amount of time where latency to enter and time spent in the center are measures of anxiety-like behavior (Gould et al., 2009; Hall, 1934; Ivinskis, 1970). The Open Field is also used to measure locomotor activity (Seibenhener and Wooten, 2015) which is often reported as movement over time. For a comprehensive review of behavioral tasks for measuring anxiety see (Lezak et al., 2017).

While the predictive validity of the EPM has been proven using anxiolytics (Pellow et al., 1985; Pellow and File, 1986), the anxiogenic effect can vary greatly based on the precise parameters used including the height of the lip on the open arms, height of the ledge from the floor, and lighting conditions (Fernandes and File, 1996; Treit et al., 1993). These parameters are often not reported, making direct comparisons between findings from different labs less than reliable. In addition, there is evidence that repeated use of the EPM is susceptible to learning effects such that time spent in and number of entries into the open arms decreases during repeated testing (Dawson et al., 1994; Fernandes and File, 1996; Treit et al., 1993). For these reasons, its utility in measuring anxiety through unconditioned avoidance is limited to a single novel session (Waif and Frye, 2007). The Open Field task is also validated with anxiolytics (Prut and Belzung, 2003) but, unlike the EPM, does not require exploration for creating a visual catalog of the apparatus and is therefore less influenced by learning effects as demonstrated by minimal effects on locomotion with repeat testing (Russell and Williams, 1973; Sturman et al., 2018; Valle, 1971). In addition, since rodents are nocturnal animals, both the Open Field and EPM can be performed under various light conditions to elicit different levels of anxiety (Ramos et al., 2002) such that bright light is more aversive than low light (Ramos and Mormède, 1997). However, when testing rodent models that exhibit high trait anxiety or under high anxiogenic conditions, it is difficult to detect differences with the Open Field and EPM as they, like many behavioral tasks, are subject to “ceiling” and/or “floor” effects.

Despite their limitations, these tests have been successfully used to measure trait and state anxiety in animal models. Several models exist that are important for understanding the genetic contribution to anxiety disorders (Finn et al., 2003; Ramos et al., 2002, 2008). One such model is the Lewis rat (Ramos et al., 2002). Lewis rats are an inbred strain known for their high trait anxiety (Ramos et al., 1997, 1998) as well as Hypothalamus-Pituitary-Adrenal (HPA) axis dysfunction (Cohen et al., 2006). Given the reliably high expression of anxiety-like behavior in Lewis rats, they can be used to test the construct validity of a task intended to measure anxiety-like behavior. In addition, a sensitive task with a wide range for detecting anxiety-like behavior is needed for animals that produce high anxiety-like behavior, particularly if they are tested under the high anxiogenic condition of bright light. Here we provide evidence that a variation of the Open Field test, the Light-Dark Open Field (LDOF) that includes components of the Light-Dark Box, yields such an increase in sensitivity with the simple addition of a shadow in the Open Field apparatus.

2. Materials and methods

2.1. Animals

Young adult (2 months old) female (175–200 g) and male (250–300 g) Sprague-Dawley (SD) rats and male Lewis rats (225–275 g) (Charles River Laboratories Inc, MA) were housed in pairs on a 12 h light/dark cycle (lights on at 7:00 am) with food and water freely available. Experiments were conducted during the light phase and were initiated no sooner than 72 h after arrival and habituation to handling. The number of rats used are listed in each figure. Animals were first run on the Light-Dark Open Field then on the EPM. Animal use protocols were approved by the Charlie Norwood VA Medical Center Institutional Animal Care and Use Committee and comply with National Institutes of Health guide for the care and use of Laboratory animals.

2.2. Handling

All rodents were handled for 3 consecutive days for 2–3 min. Behavioral testing began the day after handling for all groups. Male SD and Lewis rats were handled by one experimenter while female SD rats were handled by another. This may be a confound in the data presented however sexes were not combined and no comparisons were made between them. Animals were carried in transfer cages from their housing room to the testing room and placed into the LDOF almost immediately.

2.3. Light-Dark Open Field (LDOF) apparatus and experimental design

The LDOF (Fig. 1) is a circular arena measuring 142 cm diameter x 60 cm height. The floor of the apparatus is painted dark blue to contrast with white rats for the purpose of accurate measurements using behavioral analysis software (Noldus Ethovision XT 14). The maze arena was broken down into three areas (or zones):

Fig. 1.

Fig. 1.

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The Light-Dark Open Field. (A) Illustration of the LDOF layout. The center covers 25% of the total area of the arena. The Dark Perimeter is created with illumination from lights placed on the floor and covers 20% of the area. The Light Perimeter (20% area) opposes the Dark Perimeter. The rat is introduced to the LDOF arena as shown. (B) Illustration of Open Field parameters within the LDOF which includes the traditional zones of Total Perimeter and Center. (C) Representative picture of LDOF with shadowed Dark Perimeter.

  • Center - 25% of the total arena area and a diameter of ½ of the total diameter of the arena

  • Dark Perimeter – 20% of the total arena; a shadowed crescent-shaped area adjacent to the wall.

  • Light Perimeter - 20% of the total arena; a crescent-shaped area adjacent to the wall and opposite of the Dark Perimeter

Delta lux is defined as Center lux – Dark Perimeter lux.

The Dark Perimeter was created with flood-lights positioned on the floor to cast a shadow with sharp discrimination between light and dark. Lights in the testing room are on different switches and were selectively turned on or off to create different lighting conditions and when this was not sufficient, cloth barriers were placed over lights. The settings for lights to establish different delta lux used in these experiments is specific to this testing room and will have to be established in each testing room based on its own light configuration for replication to be possible. Lux was measured in all three zones for each light setting (Fisherbrand™ Traceable™ Dual-Range Light Meter). Animals were first introduced to the arena facing the wall at the edge of the Dark Perimeter (see Fig. 1A). This restricts their initial choice to light and dark (removing open space) and thus providing them with a simple choice point (Balleine and Ostlund, 2007; Crannell, 1942; Tolman, 1938).

All behavior was recorded and animal location was tracked using Ethovision XT 14 (Noldus Information Technology Inc). Male SD (n = 24) and Lewis (n = 24) rats were tested on the LDOF for 15 min and were used to compare strain effects in the LDOF with delta lux 120 (Center 140 lux; Dark Perimeter 20 lux; Light Perimeter 97 lux). To establish the effect of light intensity and demonstrate utility with female SD rats (n = 49), they were tested for 30 min under varying delta lux conditions. All data reported are for the first 10 min with the exception of Fig. 4 which presents data for both 10 and 15 min of habituation. In our hands, regardless of sex (data not shown) or strain, most of the habituation to the arena occurred within the first 10 min (Fig. 4). Repeat testing on the LDOF was done with a 24 h inter-trial interval. Total Perimeter, defined as 75% of the total arena, which is the traditional zone in the Open Field test, was also obtained from LDOF. The center was common to both the LDOF and Open Field analysis.

Fig. 4.

Fig. 4.

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Behavior of SD and Lewis rats in the LDOF. Male SD and Lewis rats (A) Percentage of time spent in the Dark Perimeter, Light Perimeter, and Center of the LDOF; (B) Light Anxiety Index expressed as [1 – %Time in Light Perimeter/%Time in Dark Perimeter], Open Space Anxiety Index expressed as [1–4/5%Time Spent in Center/Time Spent in Light Perimeter], and LDOF Anxiety Index expressed as [1 – 4/5%Time Spent in Center/Time Spent in Dark Perimeter]; (C) Number of rearings in the Dark Perimeter, Light Perimeter, Center, and Total rearings; (D) Representative heat map of time spent in the LDOF illustrating differences in thigmotaxis; (E) Open Field measures of time spent in the Total Perimeter and Center, taken from the LDOF. (SD n = 24, Lewis n = 24). *P < 0.001.

Three indices were developed to interpret novel data from the LDOF:

  1. The Light Anxiety Index which is [1- %Time in Light Perimeter/%Time in Dark Perimeter]. This index quantifies the extent of light aversion such that higher numbers represent increased anxiety to light.

  2. The Open Space Anxiety Index which is [1–4/5%Time in Center)/%Time in Light Perimeter]. This index quantifies the extent of open space aversion such that higher numbers represent increased anxiety to open space.

  3. The LDOF Anxiety Index is [1–4/5%Time in Center/%Time in Dark Perimeter]. This index combines both anxiogenic components within the LDOF: light and open space, with the center representing high light and open space and the Dark Perimeter representing low light and sheltered space. The LDOF Anxiety Index allows for an integrated quantification of anxiety-like behavior such that higher numbers represent increased overall anxiety.

For the purpose of these indices, it is ideal to include time spent in equivalent-size areas to reduce confounds in parsing out the effect of light and open space. Thus the Light Anxiety Index uses an equivalent area of Light Perimeter and Dark Perimeter to control for open space and maximize the contribution of light to the index. In the Open Space Anxiety Index and the LDOF Anxiety Index, %Time in Center is modified with a 4/5 multiplier to scale the area of the center to the area of the Light or Dark Perimeter.

With these indices, one can parse out the effect of two anxiogenic stimuli (light and open space) at the same time and while the rat is in the same state to determine what is driving the anxiety-like behavior. An underlying assumption is that with no anxiety rats would spend equal amounts of time across the entire area of the LDOF arena.

Behavioral measures were also examined in the LDOF. Rearings were scored when rodents put their weight on their hindlegs and raised their forelimbs and head upwards. Thigmotaxis was quantified as time spent along the perimeter while in close contact to the wall of the apparatus. Using Noldus Ethovision XT 14 an outer ring of 20% the size of the arena, slightly wider than the rodent, was defined.

2.4. Elevated Plus Maze (EPM)

The EPM is plus-shaped with four 50 cm × 12 cm arms, elevated 84 cm above the floor. Two opposite arms are surrounded on three sides by 46 cm tall opaque walls and the other two arms are open, except for a 1 cm high ledge, and brightly illuminated with approximately 200 lux in the center and approximately 5 lux in the darkest part of the closed arms. Each animal was introduced into the center area (10 cm × 10 cm) facing an open arm and allowed to explore freely for 5 min. Time spent in open arms and number of arm entries were scored; an arm entry was scored when all four paws and the base of the tail of the animal entered an arm.

2.5. Statistical methods

One-way RM-ANOVA with repeated factor Time as well as mixed design RM-ANOVA with factor Strain, repeated factor Time were used for habituation comparisons and repeated trial comparisons. One-way ANOVA with factor Strain or Delta Lux was used for between group comparisons with Fisher’s PLSD post hoc test. One sample t-test with hypothesized mean 20 (the theorized % time spent if animals spent equal time across the entire area) was used for comparisons on LDOF measures in Fig. 2B and hypothesized mean of 1 was used in Fig. 3C. Differences in proportions between groups of animals making open arm entries on the EPM were compared using the Wald-Wolfowitz run test. Outliers were determined as being more than two standard deviations from the mean and removed. Differences were considered statistically significant at p < 0.05 (StatView software, SAS Institute, Cary, NC).

Fig. 2.

Fig. 2.

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Effect of Different Lighting Conditions in the LDOF. Behavior of female SD rats in the LDOF under different illumination between the Center and Dark Perimeter (Delta Lux 5 (n = 10), delta lux 20 (n = 13), delta lux 40 (n = 11), delta lux 120 (n = 20)). (A) Table legend of delta lux and corresponding Center and Dark Perimeter illumination expressed in lux; (B) Percent time spent in the Dark Perimeter, Light Perimeter and Center, dotted line at 20 represents the theorized % time spent if animals spent equal time across the entire area. Asterisks represent significant difference from delta lux 5; (C) Open Field measures of % time spent in the Total Perimeter and Center, taken from the LDOF; (D) Light Anxiety Index expressed as [1 – %Time in Dark Perimeter/%Time in Light Perimeter]; (E) Open Space Anxiety Index expressed as [1–4/5%Time Spent in Center/Time Spent in Light Perimeter]; (F) LDOF Anxiety Index expressed as [1 – 4/5%Time Spent in Center/Time Spent in Dark Perimeter]. *P < 0.05.

Fig. 3.

Fig. 3.

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Repeat testing on the LDOF. Female SD rats under delta lux 40 light conditions; (A) Percentage of time spent in the Dark Perimeter, Light Perimeter, and Center of the LDOF over 2 trials separated by 24 h; (B) Trial 2 Light Anxiety Index expressed as [1 – %Time in Light Perimeter/%Time in Dark Perimeter], Open Space Anxiety Index expressed as [1–4/5%Time Spent in Center/Time Spent in Light Perimeter], and LDOF Anxiety Index expressed as [1 – 4/5%Time Spent in Center/ Time Spent in Dark Perimeter]; (C) Ratios of LDOF indices expressed as Trial 2/Trial 1. (n = 11). *P < 0.05.

3. Results

3.1. The Light-Dark Open Field (LDOF) task allows simultaneous assessment of anxiety to open spaces and bright light- Face validity

The LDOF allows simultaneous assessment of anxiety to bright light and open spaces. We analyzed parameters afforded to us by the LDOF (% time spent in: the Dark Perimeter; the Light Perimeter, and the Center) and calculated three anxiety indices: Light Anxiety Index, Open Space Anxiety Index, and LDOF Anxiety Index (Fig. 2). To elucidate the optimal light parameters for the LDOF, we investigated behavioral responses of SD rats to different delta lux conditions (Fig. 2A–B). Lower delta lux was created under lower light conditions with increasing ambient light intensity to create larger delta lux and thereby increase the anxiogenic influence of light. One animal from the delta lux 40 group was excluded because their %Time Spent in the Dark Perimeter was more than 2 standard deviations from the mean.

As illustrated in Fig. 2B, low light conditions with delta lux of ≤20 does not produce a strong preference for the Dark Perimeter whereas a delta lux of ≥40 does. Animals spent significantly less than 20% time, a theoretically predicted time based on even distribution of time across the area, in the Dark Perimeter for Delta lux 5 (p = 0.0072) and delta lux 20 (p = 0.0117). Conversely, animals in the delta lux 40 and 120 conditions spent significantly more than 20% time in the Dark Perimeter (p = 0.0108 and p < 0.0001 respectively). Surprisingly, SD rats appear to have a preference for the Light Perimeter compared to the Dark Perimeter at delta lux of ≤20, suggesting that there is no innate preference for the space covered by the Dark Perimeter. However, as the light intensity increases (bright light, delta lux of ≥40), their preference is changed to favor the Dark Perimeter, confirming that bright light is aversive and this is captured in the Light Anxiety Index.

When compared to the lowest anxiogenic condition of delta lux 5, rats in delta lux 40 and 120 condition spent significantly higher % time in the Dark Perimeter (p < 0.0001) and significantly lower in the Light Perimeter (p < 0.0001). In other words, under low light conditions, the rats were willing to venture into the Light Perimeter more than when the light was brighter. This is substantiated by a negative Light Anxiety Index (Fig. 2D) for delta lux of ≤20 that is significantly different from delta lux of ≥40 when examining delta lux overall effects [F(3,50) = 22.248, p < 0.0001] and post hoc tests (p < 0.0001 for 120 v 20, 120 v 5, 40 v 20, and 40 v 5).

The Open Space Index showed no difference in any delta lux condition [F(3,50) = 0.215, p = 0.8856] indicating that these rats had similarly high aversion to open space (Fig. 2E). However, the LDOF Anxiety Index (Fig. 2F), which combines both factors, is significantly higher under delta lux 120 than delta lux 20 and 5 (p = 0.0315 and p = 0.0013 respectively). There was no difference between delta lux 120 and 40 (p = 0.9452). This implies that delta lux ≥ 40 and its associated bright light is required to detect high anxiety-like behavior in this apparatus.

Open Field parameters are illustrated in Fig. 2C and show no differences between lighting conditions in % time spent in the total perimeter and the center [F(3,50) = 0.833, p = 0.4821]. From this data, one might interpret that the effect of light on anxiety among these animals is minimal but upon examining LDOF parameters, meaningful differences emerge.

Given that under higher anxiogenic conditions (bright light, delta lux ≥ 40) animals spent more time in the Dark Perimeter and overall, less time in the center than either the Light or Dark Perimeter, the LDOF data support the face validity of the task. Taken together, using the LDOF, one can detect and dissociate differences between anxiety to light, open space, and to the combined effect of light and open spaces.

3.2. Repeat testing in the LDOF

To determine the effect of repeat testing on the LDOF, we tested SD rats under delta lux 40 lighting conditions twice with a 24 h inter-trial interval. This delta lux was the lowest that created a preference for the dark perimeter.

Although there was a significant increase in the percentage of time spent in the Center on Trial 2 [F(1,10) = 9.089, p = 0.0130], there was no difference in time spent in the Dark Perimeter [F(1,10) = 4.192, p = 0.0678] or the Light Perimeter [F(1,10) = 2.623, p = 0.1364].

The LDOF indices for Trial 2 (Fig. 3B) shows anxiety to light and open space. Compared to Trial 1 delta lux 40 condition (Fig. 2D–F), on repeat testing, there is a decrease in Light Anxiety Index [F(1,10) = 13.214, p = 0.0046], Open Space Anxiety Index [F(1,10) = 8.628, p = 0.0149] and LDOF Anxiety Index [F(1,10) = 5.6839, p = 0.0383].

Because the response on repeat test was different for individual animals and to examine the effect more meaningfully, we normalized within an animal with ratio measures for each LDOF index (Fig. 3C). We compared ratios to 1 which represents no difference from trial 1. Although there was no significant difference in any of the 3 Index ratios (Light Anxiety Index p = 0.0780, Open Space Anxiety Index p = 0.1280, LDOF Anxiety Index p = 0.452), the Light Anxiety Index revealed that anxiety to light is most affected by repeat testing. However, the direction was not uniform for all animals with some animals having more anxiety and others having less anxiety to light.

3.3. Construct validity in the LDOF

To test construct validity on the LDOF we compared SD and Lewis rats on the most anxiogenic lighting condition (delta lux 120). This delta lux and light intensity was chosen as to push both strains towards ceiling effects to simultaneously test the sensitivity of the LDOF. As mentioned in the introduction, Lewis rats are an inbred strain selected for an anxiety-like phenotype with documented disruption of the hypothalamus-pituitary-adrenal (HPA) axis. Therefore, a task with construct validity should reveal that Lewis rats display more anxiety-like behavior in the LDOF under these conditions compared to SD rats. Indeed, this was our observation. The light intensity of the delta lux 120 condition was anxiogenic to both strains as they spent most of their time in the Dark Perimeter and the least time in the Center. However, Lewis rats spent significantly more time in the Dark Perimeter (Lewis = 80.32% vs SD = 40.83%) [F(1,46) = 156.485, p < 0.0001], and less time in the Light Perimeter or Center compared to SD rats [F(l,46) = 147.046, p < 0.0001; F(1,46) = 17.021, p < 0.0002] (Fig. 4A). These data confirm that LDOF has construct validity by showing that Lewis rats have a higher anxiety than SD rats induced both by light and open spaces. This point is further illustrated using the Light Anxiety Index which shows that Lewis rats have a higher aversion to light than SD rats [F(1,46) = 105.232, p < 0.0001] in addition to higher overall anxiety as measured by the LDOF Anxiety Index [F(1,46) = 17.862, p = 0.0001] (Fig. 4B). Both SD and Lewis rats have a very high Open Space Anxiety Index indicating that they are both highly averse to open space but a trend exists such that Lewis rats are more anxious to open space than SD rats (p = 0.0569).

Behaviorally, SD rats engaged in significantly more exploration of the arena than Lewis rats in all zones as measured by rearings (Fig. 4C). Examination of thigmotaxis showed a similar distribution as Fig. 4A such that Lewis rats displayed more thigmotaxis and mostly in the dark perimeter (Fig. 4D – graph not shown), further demonstrating that Lewis rats have a higher anxiety-like phenotype.

The LDOF can be used to extract parameters of a traditional Open Field task. Lewis rats spent significantly less time in the center [F(1,46) = 17.021, p < 0.0002] and more time in the Total Perimeter [F(1,46) = 50.935, P < 0.0001] of the Open Field compared to SD rats (Fig. 4E). Although statistically significant, the differences are effectively very small (time spent in the Total Perimeter Lewis Mean = 99.584% compared to SD mean = 96.980%) and can be missed if group sizes are small and comparisons have decreased statistical power.

Fig. 4 shows measures of the LDOF and Open Field highlighting the utility of the LDOF in providing measurable behaviorally-meaningful differences, such as stark Light Anxiety Index differences, which could normally be obscured by ceiling effects. Additionally, it illustrates the relationship between accepted anxiety-like behavior (low rearings in the center vs periphery, Fig. 4C) and time spent in the dark perimeter of the LDOF (Fig. 4A).

3.4. Light-Dark Open Field is sensitive enough to reveal strain differences in habituation

Another useful aspect of both the Light Dark Open Field and the Open Field tests is the ability to determine habituation – decrease in distance moved over time (here shown in 1-minute increments, Fig. 5). Habituation occurred over 15 min for both Lewis [F(23,14) = 20.096, p < 0.0001] and SD [F(23,14) = 7.100, p < 0.0001] rats, with a significant Strain x Time interaction [F(1,14) = 24.033, p < 0.0001]. Limiting analysis to the first 10 min still showed significant strain difference in habituation [F(1,46) = 87.880, p < 0.0001], significant Time effect [F(1,9) = 18.089, p < 0.0001], as well as a significant Strain x Time interaction [F(1,9) = 5.990, p < 0.0001]. Moreover, the majority of the habituation from the first 15 min occurred in the first 10 min (68% for SD and 92% for Lewis rats).

Fig. 5.

Fig. 5.

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Habituation Curves in the LDOF for SD and Lewis rats. Distance moved over 15 min for Male SD and Lewis rats. (SD n = 24, Lewis n = 24) showing significant decrease over 10 min and 15 min. *P < 0.0001.

Taken together, the data from the LDOF provide evidence that this is a task which measures two anxiogenic stimuli simultaneously: light and open space, to provide a novel and sensitive tool to assess anxiety to these stimuli in preclinical models.

3.5. Comparison with Elevated Plus Maze

The EPM also tests rodent anxiety using dimensions of light and open space. Strain comparison on EPM under similar bright light conditions shows no statistical difference between SD and Lewis rats on number of open arm entries [F(1,46) = 2.217, p = 0.1433] and total entries [F(1,46) = 2.507, p = 0.1202] (Fig. 6A). This indicates that bright light was sufficiently anxiogenic to both strains as to cause a ceiling effect on the EPM. Using an unconventional measure, the proportion of animals within a strain that made 1 or more entries into the open arms, Lewis rats (4.2%) was significantly lower than SD rats (16.7%) (p < 0.0001, Fig. 6B) illustrating existing strain differences that are readily missed under such bright light conditions with traditional measures of anxiety.

Fig. 6.

Fig. 6.

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Behavior of SD and Lewis rats in the EPM under bright light conditions. Male SD and Lewis rats A) Number of entries into the open arms, closed arms, and total entries on the EPM; B) Proportion of rats that made 1 or more entries into the open arms. (SD n = 24, Lewis n = 24). *P < 0.0001.

One explanation for the ceiling effect is that in addition to light and open space, the EPM incorporates fear of heights or falling, a third anxiogenic stimuli for rodents (WaLf and Frye, 2007). With three different anxiogenic stimuli, it would be challenging to parse out the contribution of each stimulus to the anxiety-like behavior of the rodent as compared to only two (light and open space) in the LDOF.

4. Discussion

4.1. Summary

Anxiety is a complex condition that requires preclinical interrogation. Researchers use behavioral tasks as well as animal models of anxiety to answer basic science questions about anxiety (Ramos, 2008). However, under high anxiogenic conditions of bright light, detecting differences between models is difficult as these tasks are subject to ceiling and/or floor effects. It is therefore important that we develop new tasks or innovate on existing ones. The Light-Dark Open Field (LDOF) that we present here is such an innovation with increased sensitivity for detecting differences under bright light conditions in addition to capturing two approach-avoidance tasks in one experiment. It further provides an opportunity to vary light conditions to assess the anxiogenic threshold for light.

We developed face and construct validity for the LDOF. Using different lighting conditions that varied the intensity of the anxiogenic stimulus of light we found that under higher anxiogenic conditions (delta lux ≥ 40) animals spent more time in the Dark Perimeter demonstrating face validity. Additionally, there is a threshold effect of light such that light intensity that produced delta lux ≥ 40 produced anxiety-like behavior. We showed construct validity by comparing two different rat strains, one outbred (SD) and one inbred for high trait anxiety (Lewis) under the most anxiogenic condition (delta lux = 120). We found that rearings and thigmotaxis were correlated with time spent in the Dark Perimeter and these were higher in the Lewis rats. These results were achieved without sacrificing traditional measures on the Open Field including ability to measure locomotion and habituation.

As briefly mentioned in the introduction, the predictive validity of the Open Field is well established with anxiolytics (Britton and Britton, 1981; Prut and Belzung, 2003; Rex et al., 1998). Additionally, the reliability of anxiety-like behavior in the Open Field has been documented and rigorously examined (Bolivar et al., 2000; Goma and Tobeña, 1978; Ivinskis, 1970; Seibenhener and Wooten, 2015; Sturman et al., 2018). Due to the thorough nature of validation on the Open Field from its inception almost 90 years ago (Hall and Ballachey, 1932), and given that the LDOF is a modification that casts only a shadow onto an existing Open Field, we did not find it necessary to sacrifice more animals for testing predictive validity on what is effectively an already validated test.

Here we demonstrate that combining features of Light-Dark Box and Open Field anxiety behavior tasks by adding a shadow to an existing Open Field apparatus adds an additional layer of discrimination. LDOF analysis showed stark differences between Lewis and SD rats such that Lewis rats spent significantly more time in the Dark Perimeter and less time in the Light Perimeter and Center compared to SD rats. Differences in anxiety to light in the LDOF between Lewis and SD rats are not likely due to differences in vision as both are albino strains with similar eye pigmentation which can be a differentiator in visual sensitivity (Prusky et al., 2002). In addition, both strains perform well on the visual water maze test in our lab (data not shown). SD rats in the LDOF appear to be more active than Lewis rats given a significantly longer distance moved over time. However, in the first minute of exploration of the LDOF, Lewis and SD rats have similar activity levels (Fig. 6). Furthermore, there was no significant difference in the number of total entries on the EPM between strains. This suggests that strain differences in activity are task dependent or driven by anxiety and not necessarily a stagnant phenotypic difference.

In addition, we provide 3 indices for the LDOF that demonstrate that anxiety-like behavior in Lewis rats was driven more by light intensity. Parsing out the contribution of light and open space is important because it revealed that rats have different motivators for approach-avoidance (anxiety-like behavior) like humans have different sources of anxiety (e.g. social anxiety, separation anxiety, generalized anxiety). This may help with selecting or manipulating more effective and/or ethologically relevant anxiety stimuli when measuring approach-avoidance behavior.

Integrative behavioral tasks such as the LDOF are few but they do exist. One such innovation is a modification of the EPM with a light-dark box on one end of the closed arms and an open field on the other (Ramos et al., 2008) to combine three anxiety-related tasks into a single trial. Rodents are allowed to freely explore the apparatus and allow for more variety in expression of reduced anxiety. The LDOF on the other hand allows for more variety in the expression of increased anxiety.

4.2. The LDOF offers several advantages

Firstly, the LDOF offers the ability to simultaneously track anxiety in 2 domains: open space and light. An anxiety gradient is created with the center being the most anxiogenic (high light and open space), the Light Perimeter next (high light and protected space, which is next to a wall), and the Dark Perimeter, the least anxiogenic (low light and protected space). To quantify novel data from the LDOF we provide 3 indices which allow us to parse out the effect of different anxiogenic stimuli:

  1. The Light Anxiety Index quantifies anxiety-like behavior induced by light.

  2. The Open Space Anxiety Index quantifies anxiety-like behavior induced by open space.

  3. The LDOF Anxiety Index combines both anxiogenic components within the LDOF: light and open space and allows for an integrated quantification of anxiety-like behavior such that higher numbers represent increased overall anxiety.

Quantifying time spent in each zone may be sufficient depending on the question being addressed with the LDOF. The indices provided are optional but they are useful for demonstrating the anxiogenic effects of light, open space or their combination, since the strength of the LDOF is the combined effect of two anxiogenic stimuli.

Like the Open Field, the LDOF can accommodate different intensities of anxiogenic stimuli (i.e. light). The aversive nature of LDOF can be altered by running the experiment under different light conditions (Ramos et al., 2002). We have established that there is a threshold of difference of light intensity between the Light Perimeter and Dark Perimeter below which the anxiogenic effect of light is lost. At delta lux 40 and above (with center illumination of lux 52 and above) light has a significant effect on time spent in the Dark Perimeter. The size of the open field may also be varied to manipulate the ‘intensity’ of open space as an anxiogenic stimulus. We have also determined that the LDOF is efficacious in female rats of mixed estrus phase. Future studies will determine the effect of estrus phase on anxiety-like behavior in the LDOF.

The Light-Dark Open Field also has construct validity and reduces experiment time. Because anxiety measures are influenced by both state and trait anxiety (Saviola et al., 2020; Steimer, 2011), different tasks are more sensitive to detecting anxiety-like behavior under different conditions and with different strains such as Lewis rats. To accommodate this nuance, researchers tend to use more than one task to ensure they are capturing valid measures of anxiety-like behavior. This practice can be quite time and resource consuming. The LDOF combines two anxiety tasks therefore eliminating the need for counterbalancing tasks run in series which is a method of scientific rigor for internal experimental validity that control for task order effects (Allen, 2017; Frye et al., 2000). This reduces experiment time and number of animals needed. In addition, when researchers use multiple tasks to measure anxiety, the results may be contradictory (Pati et al., 2018); combining two anxiety-related tasks and parsing out the contributions of different anxiogenic stimuli would help researchers avoid difficult to explain results and still provide meaningful data.

In addition, the LDOF can be used for repeat testing. We repeat tested female SD rats using the delta lux 40 condition and, although our data shows that there is an increase in time spent in the center 24 h after initial testing, there were no differences in time spent in the Light or Dark perimeter. Upon examination of LDOF Indices with repeat testing revealed that, under these conditions and with this sex and strain of rat, there was a habituation effect of both Light and Open Space Anxiety with a more pronounced effect on Light Anxiety. The LDOF and its indices as a tool for examining anxiety produces even more interesting information with repeat tests. As mentioned in the introduction, tasks such as EPM, that are influenced by learning and memory, cannot be repeated to measure anxiety because novelty is a large anxiogenic component (Treit et al., 1993) and therefore the anxiogenic nature of the task decreases with repeat exposure. Repeated daily testing on the Open Field has been shown to have minimal impact on exploration (Russell and Williams, 1973; Sturman et al., 2018; Valle, 1971), particularly in the case of young animals as it has been demonstrated that daily repeated testing with aged animals results in exploration decrements (Valle, 1971) and may also be strain specific (Bolivar et al., 2000). Importantly, repeat testing on Open Field has no demonstrable impact on animal welfare (Bodden et al., 2018). Note that repeat testing can be influenced by the size of the arena, light intensity, inter trial interval, among other factors and it will be important to establish repeat testing effects using your conditions.

Like the Open Field, the LDOF is also time flexible. Once the time required to achieve habituation has been established, the length of the task can be adjusted to minimize the time spent on the task. This can reduce the amount of time needed to perform behavioral testing while achieving reliable quantification of anxiety-like behavior.

Lastly, the apparatus used for the LDOF is versatile. We used an empty water maze as an open field for this experiment, but any open field of sufficient size and area should produce similar results. Using a water maze apparatus for this experiment conserves limited resources and space as an additional apparatus is not required. If it is not practical to use such a large apparatus, adding a shadow of comparable area (20%) to an existing Open Field may yield similar results. Again, the size of the apparatus will impact the ‘intensity’ of open space as an anxiogenic stimulus.

A notable limitation is that the apparatus used for the LDOF occupies a large amount of space. This can be mitigated by storing it on its side when not in use.

4.3. Conclusion

We present the Light-Dark Open Field, a simple modification to an existing Open Field apparatus that incorporates aspects of the Light-Dark Box with the addition of a shadow. The shadow allows for increased discrimination in detecting high anxiety-like behaviors and affording the option to calculate relative contributions of bright light and open space to observed anxiety-like behavior. Using both Lewis and Sprague Dawley rats, we show that even under bright light conditions, the LDOF is sensitive enough to discriminate behavioral differences and thus enhances researchers’ toolbox for quantifying anxiety-like behavior in pre-clinical models.

Acknowledgments

We would like to thank Danielle Crethers for technical support and Dr. Alvin Terry for helpful comments on a previous version of the manuscript.

Funding

This research was supported by funding from the Department of Veterans Affairs Merit Review Awards 1I01BX001978 and I01BX00389, and National Science Foundation (NSF) 1258111.

Footnotes

CRediT authorship contribution statement

Khadijah Shanazz – Writing – original draft, Writing – review & editing, Methodology, Investigation, Formal analysis, Project administration, Visualization. Almira Vazdarjanova – Writing – review & editing, Conceptualization, Methodology, Formal analysis, Resources, Funding acquisition, Project administration, Supervision. Rebecca Nalloor – Writing – review & editing, Methodology. Rachael Dixon-Melvin – Writing – review & editing, Investigation. Kris M Bunting – Writing - review & editing, Investigation.

Declaration of Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The content presented here does not represent the views of the Department of Veterans Affairs or the United States Government.

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