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: Under deep pentobarbital anaesthesia, mice were perfused transcardially for 1 minute with 0.9% NaCl + 0.5% heparin (400 U) in H2O (15ml/min), followed by 150 ml 4% paraformaldehyde (PF) in 0.1 M phosphate buffer (PB) for fixation. Brains were collected, postfixated for 24h in 4% PF in 0.1 M PB, rinsed for one day in 0.01 M phosphate buffered saline (PBS, pH 7.4) and then kept overnight in 30% sucrose in PBS cryoprotectant at 4 °C. Brains were frozen using liquid nitrogen and stored at −80 °C until further processing. The brains were cut in 30 μm coronal sections (bregma 0.26 to −1.58) using a cryotome and stored in 4% PF at 4 °C for at least two weeks before silver staining. Brain sections were rinsed 3 × 5 min in H2O, followed by 5 × 5 min in pre-treatment solution (0.45% NaOH + 0.6% NH4NO3 in H2O) and silver impregnated for 10 min in 0.3% AgNO3 + 5.4% NaOH + 6.4% NH4NO3 in H2O. After washing 3×5 min in 0.5% Na2CO3 + 29.7% EtOH + 0.012% NH4NO3 in H2O, slices were developed for 4 min in 0.056% citric acid (C6H8O7 ċ H2O) + 0.549% formaline + 10% EtOH + 0.012% NH4NO3 in H2O (PH adjusted to 5.9), fixated for 4 min in 37.5% Sodium thiosulfate (Na2O3S2 ċ 5H2O), and finally rinsed 3×5 min in H2O. The next day, slices were mounted on glass from a 1% gelatin + 0.01% Aluin solution, dried overnight and defatted/dehydrated through respectively 100% EtOH, 100% EtOH, 70% EtOH + 30% Xylol, 30% EtOH + 70% Xylol, 100% Xylol, 100% Xylol, 100% Xylol. Glass preparations were cover slipped using DPX mountant, dried for two days and then cleaned. Digital images of lesion sites were taken using a macro lens. For each mouse, the damaged area was mapped as a 50% transparent black layer into three coronal template sections: one anterior-, one in the middle-, and one posterior of the SCN (bregma coordinates −0.22; −0.46; −0.94 respectively). From these images, the most saturated area (covering the areas damaged in all subjects) and all area covered (covering areas damaged in at least one subject) were remapped to new corresponding template sections (Figure 2).Figure 2.