Behavioural Ethograms of Treatments-/Assays-Naïve Mice on a Novel Anxiety Multitest
Authors: A. M. Umarudeen , F. Khan 
Published: May 2024

Anxiety sensitivity deficiencies of individual preclinical anxiety tests/models are a major contributory factor to the high anxiety drug discovery attrition rates and wide anxiety disorder therapeutic gap. Mitigating this factor calls for the refinement and modification of the exiting rodent anxiety tests. Use of test batteries (deploying multiple single anxiety tests at different times) and multi-testing i.e., quasi-simultaneous experimental anxiolytic screening of rodents on multiple test apparatuses physically joined together into composite units have been suggested to obliterate the idiosyncrasies, broaden the anxiety sensitivity of individual single anxiety tests – with the overall goal of improving their translational capacity. Previously, some rodent multi-tests have been invented – including the triple test consisting of light-dark (LDM), elevated plus (EPM), and open field (OFM) mazes but the ambiguity presented by the central (neutral) platform of the EPM and the monotonous nature of the OFM components often included in its design has constituted a significant drawback. These 2 maze components have been replaced in a recently invented Triple mouse anxiety test with an elevated zero maze (EZM) and a marble-burying maze (MBM), respectively. Thus, the aim of this study is to investigate the murine anxiety-inducing and anxiety sensitive property of a composite apparatus (Triple test) created by physically joining a unit each of LDM, EZM, and MBM, in that that order, via thorough fares in-between the units. This ethological evaluation was also concurrently carried out in standard single rodent anxiety tests i.e., elevated zero maze (EZMT), elevated plus maze (EPMT) and open field (OFT) tests for comparison. Groups of treatment-naive mice (n=12; equal sexes) were each exposed to Triple set 1 – with trials initiated from the LDM component (Appendix I), Triple test 2 – with trials initiated from the middle EZM component (Appendix II), Triple test 3 – with trials commenced from the MBM component (Appendix III), single EZMT, EPMT, and OFT with trials conducted adopting appropriate standard operating procedures. Trials lasted 20 minutes for the triple and 7 seven minutes for the single anxiety tests. Behavioural data were collated and analysed by descriptive statistics. Results were expressed as absolute counts, percentages or means ± S.E.M. Forty-eight (48) different primary and derived behaviours were obtained on the triple test (EZM component, 22; LDM and MBM components each, 13) (Tables 1, 2, & 3) compared with 22, 18, and 18 obtained on the single EZMT, EPMT, and OFT, respectively (Tables 3, 4, & 5). Walking, rearing/assisted rearing, and sniffing were the most frequent behaviours exhibited by the experimental subjects across all tests. These behaviours together with chewing, stretch-attend postures (SAP), grooming, and defecation/urination constituted 80-90% of all observed behaviours in each single or multi test. Marble-burying behaviour (MBE) was least frequent behaviour at 0.1 marble buried per mouse and only afforded by Triple test’s MBM. Head dip (HD) was afforded only by the triple tests, single EPM and EZM. Significantly, central platform activities and data present in the EPMT were absent in the EZMT and Triple test trials. In both single and Triple tests, the experimental mice exhibited preferences for protected over unprotected portions of test devices (Table 6). Mice spent 47.5±4.2% in the dark (covered) compartment (DC.LDM) and 16.1±2.0% in the light (open) compartment (LC.LDM) of the LDM, 20.6±2.1% in closed (walled) (CS) and 4.4±0.6% in the open (unwalled) (OS) segments of the EZM, and 11.1±1.9% in the central (open) (CZ) and 39.0±5.4% in the peripheral (near-wall) (PZ) zones of the MBM components of Triple tests. Similarly, in the single tests, they spent 84.0±1.3% in closed and 15.9±1.3% in open segments of the EZM, 59.6±2.4% in closed, 17.2±1.7% in open, and 23.3±2.5 in the neutral central portions of the EPM, and 81.6±2.0% in CZ and 18.4±2.0% in PZ of the OFM (Table 6). Lastly, experimental mice exhibited differential test time distribution and exploratory activities on the Triple tests according to the component from which trials were initiated (Table 6). Mice initiated from the EZM (middle) component (Triple test 2) recorded a near even time and activity distribution of 38.4%, 33.6%, and 28% of the test duration spent in the Triple test’s LDM, EZM, and MBM components, respectively, (Figure 1a) – with most of the animals reaching and exploring all the 3 components within 15 minutes of trial commencement. This pattern contrasts with trials initiated from either from the LDM component (Triple test 1) in which about 92.7, 5, and 2.3% of the test duration was spent by the mice on the LDM, EZM, and MBM components, respectively (Table 6). Additionally, only in half of the trials did the mice reach the second (EZM) component and only in 2 trials did they get to the third (MBM) component from the initiating maze (Data unshown). Triple test 3 trials also exhibited an uneven activity/time distribution – with experimental mice spending about 6.5, 9.5, and 84.0% of test durations on the LDM, EZM and MBM components of the composite anxiety test apparatus – with only 5 mice reaching the second (EZM) and 2 mice reaching the third (LDM) components from the starting MBM (Data unshown). In conclusion, the triple mouse test is sensitive to and capable of generating greater spectra of anxiety-related mouse behaviours than than any standard single anxiety assay. Findings also showed single acute trials initiated from the EZM component were associated with the most exploratory activity and even spread of the test duration by the mice investigated on the Triple anxiety test. It may be necessary to investigate the behavioural effect of anxiety-related drug treatments and repeated trials on mice exposed to this novel anxiety Multitest.