Improving anxiety research: novel approach to reveal trait anxiety through summary measures of multiple states

Zoltan K Varga, Diana Pejtsik, Mate Toth, Zoltan Balogh, Mano Aliczki, Laszlo Szente, Gyula Y Balla, Levente Kontra, Zsofia Eckert, Zsolt Borhegyi, Eva Mikics

Preprint posted on 6 June 2023

Anxious rodents for the better understanding of human anxiety

Selected by Nándor Lipták

Several locomotion-based tests have been developed to assess anxiety-like behavior in rodents, such as the elevated plus maze (EPM, evokes fear of heights and open space), open-field (fear of open space) and the light-dark box (fear of light). Those tests have proven to be very useful for translational research in the past decades. However, if we want to combine or repeat those tests with the same animals in order to model patients’ chronic anxiety (“trait anxiety”), our data will not be consistent and, most importantly, will not be in harmony with the human data (Cnops et al., 2022). Anxiolytics, such as benzodiazepines or chlordiazepoxide lose their effect in rodents after repeated tests due to a phenomenon which is known as one-trial tolerance (File, 1990; File and Zangrossi,1993). The underlying mechanisms are not completely understood, and thus a novel paradigm should be introduced.

In this preprint, a battery of behavioral tests were applied to develop a better approach for studying trait anxiety in mice and rats. In addition, the whole transcriptome of the medial prefrontal cortex (mPFC) was also studied using RNASeq analysis.

Experimental design:
Female Wistar rats and male C57/BL6J mice were tested during a 3-week period with EPM, open-field and light-dark box. Each test was repeated three times for all animals. Open arm time % (OAT%, EPM), center time % (open-field) and light time % (light-dark box) parameters were measured. The first test was defined as single measures (SiMs), while the average of the scaled variables of 2 and 3 repeats of each test type were defined as summary measures (SuMs). Blood corticosterone levels of Wistar rats (baseline vs stress-induces samples) were also measured, as the end-product of the activation of the hypothalamus (CRH/AVP)-pituitary (ACTH)-adrenal cortex (corticosterone)-axis (HPA). Whole transcriptome-analysis, using RNASeq was performed on the homogenized mPFC tissues, obtained after the last tests (single end RNA sequencing, Illumina NextSeq500 sequencer).

Key findings:

Test-retest decrease was obvious in the open-arm time % in EPM. Center and light time % of the open-field and light-dark box, respectively in male and female rats, were much more consistent compared with EPM (Fig.2A). The light-dark box assay was the most reliable test of the three (Fig.2B upper). SuMs of high complexity (2 or 3 repeats of 2 or 3 tests) correlated stronger between tests than less complex SuMs or the SiMs in both species, according to the statistics (Fig.3A,3B, Table 1).

In line with the behavioral data, elevated blood corticosterone levels were observed in rats after the anxiety-inducing tests (Fig 4C). In the case of three clusters, SuMs were correlated with the mPFC RNA Seq results. Cluster 1 genes: cardiac function, cellular localization and transport, and potassium signaling. Clusters 2 and 3 genes: roles in plasticity and transcription, respectively. Only Cluster 4 genes (metabolism, stress response, and hydrolase activity-associated functional terms and unites) were SiM-associated.

Why I liked this preprint

Classic behavioral tests along with corticosterone measurement were combined with up-to-date transcriptomics and bioinformatics.

Questions for the authors

1. One-trial tolerance was the most evident in the EPM data. This issue was also studied by (Tucker and McCabe, 2017). They found that elevated zero maze is more consistent in repeated trials than EPM in C57/BL6J mice. Would elevated zero maze be more suitable for your novel trait anxiety assay than EPM?

2. Novel anxiety-like behavior tests or combinations of tests are supposed to be validated with the administration of known anxiolytics (e.g. chlordiazepoxide, benzodiazepines) in rodents. Are you planning to validate your results with anxiolytics?

3. Besides OAT%, open arm entries (OAE %) is an accepted parameter for assessing anxiety-like behavior in rodents. Same analogy for the open-field, with the center time % and center ambulation distance % parameters. Were those other two parameters (OAE% and center ambulation%) in harmony with the parameters which were highlighted in the preprint? Have you detected any species-specific differences in OAE% or center ambulation% ?

4. EPM, open-field and light-dark tests are very sensitive to any significant changes in locomotion. Have you observed significant changes in total distance traveled (open-field) or total number of entries (EPM) in mice or rats?

5. mPFC has a prominent role in regulating anxiety indeed; however, other brain regions, such as the amygdala and hypothalamus are a part of this circuit as well. Moreover, amygdala may act as a switch between repeated EPM trials in rats (Albrechet-Souza et al.,2008). Which other anxiety-controlling brain regions should be analyzed with RNASeq in rodents after your novel battery of tests?


Sandra E. File: One-trial tolerance to the anxiolytic effects of chlordiazepoxide in the plus-maze. Psychopharmacology 100 (2), 281-282, 1990.

Sandra E. File, Helio Zangrossi: “One-trial tolerance” to the anxiolytic actions of benzodiazepines in the elevated plus-maze, or the development of a phobic state? Psychopharmacology 110, 240-244, 1993.

Vanja Cnops, Vinaya Rajagopal Iyer, Nageiswari Parathy, Peiyan Wong, Gavin S Dawe: Test, rinse, repeat: A review of carryover effects in rodent behavioral assays. Neuroscience & Biobehavioral Reviews, 104560, 2022.

Tucker and McCabe: Behavior of male and female C57BL/6J mice is more consistent with repeated trials in the elevated zero maze than in the elevated plus maze. Frontiers in behavioral neuroscience 11, 13, 2017.

Lucas Albrechet-Souza, Karina G Borelli, Marcus L Brandao: Activity of the medial prefrontal cortex and amygdala underlies one-trial tolerance of rats in the elevated plus-maze. Journal of neuroscience methods 169 (1), 109-118, 2008.

Tags: anxiety, behaviour, mice, rats, tests

Posted on: 23 June 2023


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