Research report
Circadian modulation of learning and memory in fear-conditioned mice

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Abstract

Endogenous processes referred to as circadian oscillators generate many of the daily rhythms in physiology and behavior of a variety of animals including humans. We investigated the possible circadian regulation of acquisition, recall and extinction in two strains of mice (C-57/6J and C-3H). Mice were trained in either the day or night with a tone and context fear conditioning protocol. The mice were then tested over the course of several days for their ability to recall the training. When comparing the performance of animals in the day and night, the mice acquired the conditioning faster in the day than in the night. Furthermore, the recall for context and tone consistently peaked during the day for at least 3 days after training, irrespective of the time of training. Finally, the loss of this training (or extinction) exhibited a rhythm in that mice trained in night exhibited a greater degree of extinction than mice trained in the day. For all of these rhythms in acquisition, recall, and extinction the phase of the rhythm was controlled by the prior light–dark (LD) cycle. When we reversed the phase of the LD cycle, the phase of the rhythm also reversed. Importantly, all three of the rhythms also continued in constant darkness demonstrating the endogenous, and presumably circadian nature, of the rhythms.

Introduction

Most organisms, including humans, exhibit daily rhythms in their behavior and physiology. The physiological system responsible for these rhythms is known as the circadian system and, in mammals, the core of this rhythm generating system can be localized to a site in the hypothalamus known as the suprachiasmatic nucleus (SCN; [50], [59]). The endogenous rhythms generated by cells in the SCN repeat with a frequency close to, but not quite equal to, the 24-h period [2], [23], [64]. In general, the circadian timing system is thought to function to allow the temporal coordination of various physiological processes within an organism as well as allow the temporal coordination of the organism with the external world. In order to fulfill these functions, these rhythms must be synchronized to the exact 24-h cycle of the physical world. The daily cycle of light and dark is the dominant cue used by organisms to synchronize their biological clocks to the environment. Within an organism, the circadian system modulates many physiological processes and behaviors [39], [65].

Though not extensively studied, there is evidence that diurnal variation may be a general feature of performance on learning and memory tasks. Certainly animals can form associations between the time of day and food availability. In an early study, Beling [5] noted that if bees were offered sugar water at a particular time of day, they quickly learned to arrive at the feeder in anticipation of the food reward. If the sugar water was omitted, the trained bees still arrived at the feeder at the correct time. This type of time–place association has been described in many species including birds, insects, fish and mammals [8], [12], [32], [46], [47], [48], [49], [55], [62]. Other early studies on circadian phase dependence of learning have shown that animals can somehow ‘time-stamp’ information such that it is processed better at certain times of the day, where for example the performance of rats peaked at 24-h intervals following training on an avoidance task (e.g. [34]). Besides this diurnal periodicity, at least two other pieces of evidence link these rhythms in performance with the circadian system described above. First, lesions of the rat SCN [58] eliminate the 24-h rhythm in performance on a passive avoidance task. Second, Devan et al. [21] reported that rats subjected to desynchronization of the circadian system by rapidly changing the phase of the light–dark (LD) cycle experienced impaired recall of a spatial task. In addition to behavioral studies, electrophysiological studies using long-term potentiation (LTP), an electrophysiological analogue of learning and memory, has also been shown to undergo diurnal variation. For example, Barnes et al. [3] showed that synaptic responses in hippocampal granule cells following stimulation of afferent fibers from the entorhinal cortex fluctuates with a 24-h period. This group reported that synaptic activity for rats was highest in the middle of the dark phase and lowest in the middle of the light phase while the converse was true for diurnal squirrel monkeys. In another study, the magnitude of LTP, as a percentage of pretetanus basal response, was shown to vary in CA1 and dentate gyrus of hippocampal slices prepared from rats in the day or night [31]. More recently, Raghavan et al. [54] also reported diurnal variations in the magnitude of LTP in CA1 region of hippocampal slices prepared from the hamster.

In light of these studies, we designed a series of experiments to explore circadian modulation of learning and memory using a fear-conditioning paradigm. Fear conditioning is an associative-learning task that has become one of the leading behavioral models for investigating the neurobiological basis of learning and memory. Animals can learn to associate an initially neutral or conditioning stimulus (CS) such as a tone or context with a biologically significant event such as an unconditioned stimulus (US) like a footshock. In this assay, fear is measured as inactivity or ‘freezing’ after the stimulus. Freezing is a typical defensive response in rodents following exposure to aversive stimuli. The circuitry in fear-conditioning learning involves transmission of information about the CS (tone and context) and US (footshock) to the amygdala and the subsequent fear-response is linked to output projections from the amygdala to autonomic and behavioral responses in the brainstem [1], [37]. In some experimental conditions, context, but not tone, learning requires normal hippocampus function [36], [41], [63].

In the present study, we investigated the possible circadian regulation of acquisition, recall and extinction in two strains of mice (C-57/6J and C-3H) following fear-conditioning training. The use of these two strains allows a comparison between the C-3H strain that secrete melatonin rhythmically and the C-57 strain that does not [22], [30]. Animals were trained either during the day or at night. Mice were subsequently tested at least 24 h after training for context and tone learning every 6 h for 3 days. The degree of acquisition was greater in animals trained during the day then in animals trained at night. On tests for context and tone both strains of mice showed daily rhythms in recall, where greatest recall was usually observed when animals were tested during the day. Finally, the degree of long-term extinction of memories for context and tone were also shown to vary from day to night. Each of these diurnal rhythms persisted when animals were maintained in constant darkness, a finding that demonstrates the endogenous nature of these rhythms.

Section snippets

Subjects

Two-month old male mice (C-57/6J and C-3H) were purchased from Charles Rivers Laboratories. The UCLA Animal Research Committee approved the experimental protocols used in this study. Animals were housed in cages, which were placed in light-tight chambers where the light cycle could be controlled. For each experiment 8 mice were trained in the day and another 8 mice were trained at night.

Experimental procedure

Mice were allowed to entrain to the required LD cycle for at least 1 week prior to training on the

Rhythm in acquisition of C-3H mice

Our first experiment was designed to determine whether the ability of C-3H mice to learn the fear conditioning protocol varied between day and night. Mice trained in the day were trained 3 h after lights-on (ZT 3; Fig. 1a) while mice trained at night were trained 3 h after lights-off (ZT 15; Fig. 1b). Other than the time that the animals were trained and tested, all other conditions between the day and night groups remained constant. The degree of acquisition (Fig. 2a) was greater in mice

Discussion

In the present study, C-3H and C-57 mice were trained with a context and tone fear conditioning protocol. The mice were then tested over the course of several days for their ability to recall the training. When comparing the performance of animals during the day and night, the mice acquired the conditioning faster in the day than in the night. Furthermore, the recall for context and tone consistently peaked during the day for at least 3 days after training, irrespective of the time of training.

Conclusion

The data presented here show that different aspects of memory, namely acquisition, recall and long-term extinction for simple associative memory in mice is modulated by the circadian system. Since learning and memory function is based on biological processes and most biological processes are rhythmic, it should not be surprising that circadian rhythms were seen in acquisition and recall of learned behaviors. It may be that these daily rhythms represent a bi-product or epi-phenomena of a

Acknowledgments

Supported by Whitehall Foundation grant #F98P15, NIH #HL64582 and MH59186. We are also grateful to Dr M.S. Fanselow (University of California, Los Angeles, USA) whose laboratory generously aided in the collection of the preliminary data upon which this study was based.

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