Neither the SCN nor the adrenals are required for circadian time-place learning in mice.
Bottom Line: During Time-Place Learning (TPL), animals link biological significant events (e.g. encountering predators, food, mates) with the location and time of occurrence in the environment.Abrupt FEO phase-shifts (induced by advancing and delaying feeding time) affected TPL performance in specific test sessions while a LEO phase-shift (induced by a light pulse) more severely affected TPL performance in all three daily test sessions.We conclude that, although cTPL is sensitive to timing manipulations with light as well as food, neither the SCN nor the adrenals are required for cTPL in mice.
Affiliation: Department of Molecular Neurobiology and.
During Time-Place Learning (TPL), animals link biological significant events (e.g. encountering predators, food, mates) with the location and time of occurrence in the environment. This allows animals to anticipate which locations to visit or avoid based on previous experience and knowledge of the current time of day. The TPL task applied in this study consists of three daily sessions in a three-arm maze, with a food reward at the end of each arm. During each session, mice should avoid one specific arm to avoid a foot-shock. We previously demonstrated that, rather than using external cue-based strategies, mice use an internal clock (circadian strategy) for TPL, referred to as circadian TPL (cTPL). It is unknown in which brain region(s) or peripheral organ(s) the consulted clock underlying cTPL resides. Three candidates were examined in this study: (a) the suprachiasmatic nucleus (SCN), a light entrainable oscillator (LEO) and considered the master circadian clock in the brain, (b) the food entrainable oscillator (FEO), entrained by restricted food availability, and (c) the adrenal glands, harboring an important peripheral oscillator. cTPL performance should be affected if the underlying oscillator system is abruptly phase-shifted. Therefore, we first investigated cTPL sensitivity to abrupt light and food shifts. Next we investigated cTPL in SCN-lesioned- and adrenalectomized mice. Abrupt FEO phase-shifts (induced by advancing and delaying feeding time) affected TPL performance in specific test sessions while a LEO phase-shift (induced by a light pulse) more severely affected TPL performance in all three daily test sessions. SCN-lesioned mice showed no TPL deficiencies compared to SHAM-lesioned mice. Moreover, both SHAM- and SCN-lesioned mice showed unaffected cTPL performance when re-tested after bilateral adrenalectomy. We conclude that, although cTPL is sensitive to timing manipulations with light as well as food, neither the SCN nor the adrenals are required for cTPL in mice.
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Mentions: The used TPL test apparatus and testing procedures were described before (Mulder et al., 2013c; Van der Zee et al., 2008). Briefly, to induce food-seeking behavior and voluntary location-choices, mice were food deprived to 85% of their ad libitum body weight, as individually determined by the average of three daily measurements prior to initiating food deprivation. To monitor bodyweight during testing, mice were weighed before being tested in each daily session and received an individual amount of food at the end of the light-phase (ZT10.5). Homecage control (HCC) mice were not TPL tested, but similarly food deprived (unless stated otherwise). Mice were tested in their inactive (light-) phase. In each of three daily sessions (lasting maximally 10 minutes per mouse), TPL test mice had to learn to avoid one of the three presented feeding locations (bated with powdered standard rodent chow, <0.1g), depending on the TOD (i.e. session). On visiting the non-target location, mice received a mild but aversive foot-shock (set to 620 volts; 0.09 mA; <1 s). A session was considered correct, on an individual level, only when the two target locations were visited first, avoiding the non-target location or visiting it lastly. Daily performance was calculated for each animal as the percentage of correct sessions (e.g. 0, 33, 67 or 100%) and these performances were averaged and plotted per group, forming a learning curve over multiple testing days. Mice from the two groups were alternated in the testing sequence. Actual testing was preceded by habituation steps as described previously (Mulder et al., 2013c; Van der Zee et al., 2008). See supplemental data in Van der Zee et al. (2008), for a graphical representation of the habituation steps. In short, target locations were always baited. During the first four days (1–4), the non-target location was also baited so that all locations were safe to explore freely (no foot-shock delivery). During the next three days (5–7), the non-target location was kept unbaited, but still safe to visit (no foot-shock delivery). During the following three days (8–10), the shock was introduced at the non-target location, while still kept unbaited, so that mice could identify the non-target location based on sight and smell. On day 8, mice were habituated to first-time foot-shock exposure. The non-target location was kept inaccessible until the mice had first consumed the food rewards in the two target locations. This way, in each session all mice received both the positive food experience, followed by the negative foot-shock experience. Because of the manipulation, day 8 was excluded from further analysis. After these habituation steps, actual testing started with all locations baited and foot-shock delivery in the non-target location. Hence, mice could not identify the non-target / target location(s) based on sight/smell and had to use knowledge of circadian phase to discriminate the hazardous non-target location. A schematic overview of the daily protocol is provided below (Figure 1).Figure 1.