The present study aimed to establish the effect of time of the day on habituation in the open-field test, one of the most elementary forms of non-associative hippocampal-dependent learning. Open-field test was performed in young adult male Wistar rats at the beginning (08:30-10:00 h; defined as Zeitgeber time (ZT) ZT0.5-2), mid-time (13:00-14:30 h, ZT5-6.5) and at the end (18:30-20:00 h, ZT10.5-12) of the light period. Our results revealed that in the acquisition trial there were no significant differences among the six parameters recorded through tested periods. In contrast, the level of habituation in the ambulation and rearing rose as followed: ZT0.5-2 < ZT10.5-12 < ZT5-6.5. In both trials, the principal component analysis highlights two components: component 1 was mainly loaded by ambulation in the outer and inner area, rearing and freezing behaviors, whereas component 2 was mostly loaded on grooming activity and defecation. The correlation between parameters varied across the period of day and trial. Animals that expressed a higher level of grooming, defecation (ZT5-6.5) and freezing behavior (ZT0.5-2 and ZT5-6.5) at acquisition trial habituated better on those parameters on the retention trial. In conclusion, habituation outcomes to the open-field test and correlation between tested parameters highly depend on daytime.
Keywords: Anxiety; Habituation; Open-field test; Rat; Time-of-day.
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