Caroline H. Ko

The genetics of mammalian circadian order and disorder: implications for physiology and disease

Circadian cycles affect a variety of physiological processes, and disruptions of normal circadian biology therefore have the potential to influence a range of disease-related pathways. The genetic basis of circadian rhythms is well studied in model organisms and, more recently, studies of the genetic basis of circadian disorders has confirmed the conservation of key players in circadian biology from invertebrates to humans. In addition, important advances have been made in understanding how these molecules influence physiological functions in tissues throughout the body. Together, these studies set the scene for applying our knowledge of circadian biology to the understanding and treatment of a range of human diseases, including cancer and metabolic and behavioural disorders.

Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes

The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and although rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism and insulin signalling is delayed in circadian mutant mice, and both Clock and Bmal1 (also called Arntl) mutants show impaired glucose tolerance, reduced insulin secretion and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival and synaptic vesicle assembly. Notably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective β-cell function at the very latest stage of stimulus–secretion coupling. These results demonstrate a role for the β-cell clock in coordinating insulin secretion with the sleep–wake cycle, and reveal that ablation of the pancreatic clock can trigger the onset of diabetes mellitus.

Intercellular Coupling Confers Robustness against Mutations in the SCN Circadian Clock Network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.

Dissecting the Functions of the Mammalian Clock Protein BMAL1 by Tissue-Specific Rescue in Mice

The basic helix-loop-helix (bHLH)–Per-Arnt-Sim (PAS) domain transcription factor BMAL1 is an essential component of the mammalian circadian pacemaker. Bmal1–/– mice lose circadian rhythmicity but also display tendon calcification and decreased activity, body weight, and longevity. To investigate whether these diverse functions of BMAL1 are tissue-specific, we produced transgenic mice that constitutively express Bmal1 in brain or muscle and examined the effects of rescued gene expression in Bmal1–/– mice. Circadian rhythms of wheel-running activity were restored in brain-rescued Bmal1–/– mice in a conditional manner; however, activity levels and body weight were lower than those of wild-type mice. In contrast, muscle-rescued Bmal1–/– mice exhibited normal activity levels and body weight yet remained behaviorally arrhythmic. Thus, Bmal1 has distinct tissue-specific functions that regulate integrative physiology.

Emergence of Noise-Induced Oscillations in the Central Circadian Pacemaker

Computational modeling and experimentation explain how intercellular coupling and intracellular noise can generate oscillations in a mammalian neuronal network even in the absence of cell-autonomous oscillators.

Circadian rhythms, aging and memory

In human beings and animal models, cognitive performance is often impaired in natural and experimental situations where circadian rhythms are disrupted. This includes a general decline in cognitive ability and fragmentation of behavioural rhythms in the aging population of numerous species. There is some evidence that rhythm disruption may lead directly to cognitive impairment; however, this causal link has not been made for effects due to aging. We have tested this link by examining rhythms and performance on contextual conditioning with the conditioned place preference task, in elderly, age-matched hamsters. Young healthy hamsters developed a preference for a context that is paired with the opportunity to engage in wheel-running (experiment 1). Aged animals with consolidated locomotor rhythms developed similar degrees of preference, whereas the age-matched hamsters with fragmented rhythms did not (experiment 2). The degree of preference was also correlated with activity amplitude. These results support the notion that age-related rhythm fragmentation contributes to the age-related memory decline.

The significance of circadian phase for performance on a reward-based learning task in hamsters.

In humans and animal models, circadian modulation of learning has been demonstrated on numerous tests. However, it is unclear which aspects of the cognitive process are rhythmically regulated. In these experiments, we used a conditioned place preference task in hamsters to ask whether memory acquisition (hypothesis 1) or memory recall and performance (hypothesis 2) were subject to circadian modulation. In golden hamsters, access to a running wheel has been used as a reward to condition a place preference, but when given unrestricted access to a wheel, animals perform most of their spontaneous running within a few hours each day or circadian cycle. This suggested that either the perceived reward value of the wheel changes through the day or that the response to this reward is temporally restricted. Contrary to the hypotheses, we found that learning was not tied to the time of training nor to the time of testing, but rather animals showed a preference for a reward-paired context only at the circadian time that training had taken place. Timing is not an explicit discriminative cue in these experiments. Hence, the learning mechanism must be predisposed to register circadian time as an attribute during context learning.

Time of day modulation of conditioned place preference in rats depends on the strain of rat used

In golden hamsters, the expression of a conditioned place preference (CPP) or avoidance (CPA) is regulated in a circadian pattern such that the preference and avoidance are exhibited strongly at the circadian time of prior training, but not at other circadian times. In the rat, reports are conflicting regarding whether time of day learning is evident. We investigated whether this conflict arises because different strains of rat have been used. In this experiment, Long Evans and Wistar rats were trained at a specific circadian time to discriminate between a context paired with food reward and an unpaired context. Animals were then tested for preference at the same or a different circadian time. Long Evans rats showed preference for the paired context at both times tested, whereas Wistar rats showed preference only when training and testing times matched. The results show that time of day learning can be generalized to rats using the Wistar strain.

Attenuation of context-specific inhibition on reversal learning of a stimulus-response task in rats with neurotoxic hippocampal damage.

Rats with hippocampal or sham lesions were trained on a stimulus-response task developed for the 8-arm radial maze. After reaching a stringent learning criterion, different context manipulations were performed. In Experiment I, the different groups were transferred to an identical radial maze in a different room to determine the context specificity of the discrimination learning. Experiment I revealed that although rats with hippocampal lesions did not show a normal context detection effect, the expression of the discrimination was not context dependent for either the lesion or sham groups. In Experiment II, animals were trained to criterion on the discrimination task and then both groups were divided into sub-groups based on whether they would experience reversal training in the same or different context from original training. Experiment II indicated that animals with hippocampal lesions and shams reversed in a different context were significantly enhanced in reaching the learning criterion compared to either counterparts that were reversed in the same context. Reversal learning in rats with hippocampal lesions was faster than sham animals in the same context suggesting that the context-specific inhibition effect was hippocampal-based. After learning the reversal task, the groups of animals trained and reversed in different contexts were brought back into the original training context to test for competitive effects. Animals with hippocampal lesions that were reversed in the different context, did not show a competition between the most recently acquired discrimination and a context-specific association acquired during original training whereas sham animals in the same condition did. Taken together these results suggest that rats with hippocampal lesions do not acquire normal context-specific inhibition during discrimination learning.

The Suprachiasmatic Nucleus is not Required for Temporal Gating of Performance on a Reward-based Learning and Memory Task

In hamsters, the expression of a learned preference for context depends upon a temporal match between the time of training and testing. In the present experiments, we investigated the role of the biological clock in the suprachiasmatic nucleus (SCN) as a provider of temporal information underlying this time dependent modulation of cognitive performance. Hamsters were tested using the conditioned place preference task (CPP) before and after ablation of the SCN. Arrhythmic animals continued to show time-of-day modulation of the CPP when trained and tested in the absence of the SCN. This supports the notion that time of day information is a component of context representation for the hamster (Antoniadis et al., 1999), and indicates that an oscillator outside of the SCN is responsible for time discrimination in reward-based learning.