Habituation time and other standardisation parameters for select behavioural tests in Flinders Sensitive Line rats
With the aim of refining experimental procedures for animal behavioural tests commonly performed in our laboratory, this study aimed to provide empiric-evidence for specific aspects of the methodological approach of select behavioural tests and to provide direction for future research in this laboratory which may contribute to also facilitate more robust between-study comparisons. As such, the present investigation had two main objectives, viz. A) to study the effect of pre-test habituation on the performance of test subjects in a sequence of cognitive and behavioural tests performed on a single dark cycle in the same test subjects, i.e. 1) the novel object recognition test (nORT; declarative memory), the open field test (OFT; locomotor activity) and the forced swim test (FST; behavioural despair), and B) to measure how habituation will influence the treatment response of FRL and FSL rats subjected to the procedures highlighted in A to a positive control, i.e. imipramine, 10 mg/kg/day x 14 days. A secondary objective was to study the influence of pre-test habituation on the performance of test subjects if the same tests as applied in (A) were to be performed only one per day on successive days, albeit also in both treatment conditions. In this regard, for objective A, male FSL (n = 48) and FRL (n = 48) rats were employed, divided into the following groups (n = 12 per group): a) treatment naive (saline-treated) FRL rats, b) imipramine-treated FRL rats, c) treatment naive (saline-treated) FSL rats, and d) imipramine-treated FSL animals. Due to the fact that the secondary objectives could not have been completed, neither the outline for this objective, nor its results will be outlined here (see Annexure B). In the nORT, declarative memory was not affected by the two different pre-test habituation protocols, indicating that the pre-test emotional state of the animal does not significantly alter their inherent, non-manipulated cognitive performance as assessed in terms of declarative memory. With regards to OFT behaviour, alterations in locomotor activity was only apparent after 60-min pre-test habituation, resulting in FSL rats covering less distance compared to their FRL counterparts. Non-habituated FRL and FSL rats covered similar distances during the 5-min test session. In the FST, male FSL rats displayed increased depressive-like behaviour and decreased escape-related behaviour (irrespective of pre-test habituation time) compared to FRL rats, underlining the face validity of the FSL model. Both strains displayed increased depressive-like behaviour when pre-test habituation was negated, with no significant alterations in active behaviours. IMI-treated FSL rats displayed comparable cognitive and depressive-like behaviour to SAL treated counterparts in the nORT and FST, respectively. Further, IMI-treated FRL rats also displayed comparable cognitive function compared to SAL treated cohorts. However, IMI-treated FRL rats presented with exaggerated depressive-like behaviour in the FST compared to SAL receiving FRL controls. In conclusion, we have shown that the cognitive performance of both FRL and FSL rats in the nORT are robust enough to withstand varying pre-test circumstances, despite alterations in locomotion after 60-min pre-test habituation. Further, depressive-like behaviour is bolstered in both strains when tested directly after relocation, without significantly affecting active behaviours. Therefore, collectively viewed, we argue that in order not to misinterpret the behaviour of FSL animals in the FST based on findings from the OFT, albeit falsely so, both FRL and FSL animals should be subjected to both the OFT and the FST without prior habituation. Due to the confounding results from IMI receiving cohorts, the predictive validity of this model could not be re-affirmed. However, as our data contradicted the majority of previous reports, it is unlikely that these findings were borne from inherent confounds in the model. Nonetheless, valid conclusions can still be made based on the robust baseline face validity of the FSL model that has been affirmed in the present work.
- Health Sciences