How do neural microcircuits and networks in the suprachiasmatic nucleus encode circadian time?

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Common experience tells us that we are 24h beings. Most obviously we sleep and wake on a daily cycle, and over the course of day and night our emotional state, cognitive abilities and general energy levels wax and wane on a regular programme. These rhythms are cued to the timing of sunrise and sunset, but when we travel between time-zones or work on rotating shift schedules we become aware that they are also dependent on internal cues. Indeed evolution has furnished us and almost all types of living organism with internal biological clocks that control our daily cycles, and are cued by sunlight. In mammals the major internal clock is the suprachiasmatic nucleus (SCN) of the brain's hypothalamus. It received nerve input from the eye to cues it to the cycle of dark and light but it does not need this input to generate its own cycles of neuronal activity which run with a period of approximately (circa-) one day (-dian): hence it is a "circadian" clock. One of the major advances in circadian biology over the past 20 years has been the identification of the genes and proteins that make up the circadian timer. In each cell of the SCN, these genes and the proteins that they encode are locked into a self-sustaining feedback loop of protein synthesis and followed by protein degradation. The biochemistry of these process means that the loop is completed approximately once a day. It is a cellular circadian mechanism that drives parallel circadian cycles of neuronal activity in the SCN, and these cycles of neuronal activity in turn communicate daily time to the rest of the brain and body. Despite this remarkable breakthrough in unravelling the "nuts and bolts" of the cellular clockwork, it remains many fundamental questions about the SCN unanswered. For example, the SCN consists of 20,000 cells and the clockwork in each is tightly coupled to that of the others. This is a necessary property if the SCN is to give a single unambiguous time signal to the body - but how is it achieved? Second, although they are synchronised, the clocks of the individual cells are not simultaneously active. When neuronal activity in the SCN cultured as an "organotypic" slice in a Petri dish is imaged microscopically, it can be seen that waves of activity sweep across the tissue on a daily basis. This wave has a very stable and stereotypical style, common to all SCN slices. It therefore represents a conserved internal structure to the circuit, but what it is for is not clear, although some studies suggest that it may encode seasonal changes in daylength. We wish to understand how it is generated, and then we shall use that knowledge to control it using genetic and pharmacological approaches and thereby test its function. To achieve our overall aim of understanding how the SCN circuit works, we first of all need to simplify our analysis and focus on the circadian properties of particular sub-populations of SCN cells. We shall then see how cells that are connected (i.e. organised into microcircuits) behave in relation to each other. To achieve this we shall use state-of-the-art real-time microscopic methods combined with genetic targeting of cell populations that we have developed over recent years. This will allow us to follow over several circadian cycles in single SCN slices cellular rhythms of electrical activity, calcium levels (a particularly important signalling pathway for coupling) and circadian gene expression. We shall focus on microcircuits where cells are connected by signalling by excitatory neuropeptides or the inhibitory amino acid GABA. We see shall then manipulate the cellular cycles and/ or peptidergic and GABA signals to see what happens to the particular microcircuit and to the overall network. This proposal will greatly advance our understanding of the neural basis of circadian timing in mammals and should also serve as an exemplar for the analysis of how neural circuits control behaviour.