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

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.

Circadian rhythmicity in mammals is maintained by an intracellular transcriptional-translational feedback loop, whereby PER and CRY proteins negatively regulate their own expression. This oscillation is present in the majority of cells in major organs, which are in turn synchronised by a central pacemaker: the suprachiasmatic nucleus (SCN). The SCN is entrained to the light/dark cycle by retinal innervation but in organotypic slices it will indefinitely maintain a ca. 24h period, which can be monitored in real-time using bioluminescent or fluorescent reporters of PER/CRY activity. Robust clock function depends not only on cell-autonomous time-keeping, but also the emergent network-level properties of the SCN conferred by intercellular signalling. These include the tight coupling of SCN cellular rhythms, and the processive spatiotemporal waves of gene expression, intracellular calcium, and membrane potential that move across the SCN circuit. The overall objective of this proposal is to understand how the SCN operates as a circuit. How do these circuit-level properties arise as a result of interplay between cell-autonomous properties, local microcircuit-based activities and neuropeptidergic and GABAergic signalling? We shall employ selective targeting of SCN cell populations and local microcircuits using conditional expression of state-of-the-art molecular tools, first to facilitate spatiotemporal imaging and analysis of local and network functions, and, second, to confer spatiotemporal control of the network elements and identify causal mechanisms. We have developed the concept of microcircuits as an organisational feature of the SCN from our work on neuronal VIP signalling and astrocytic glutamatergic signalling. It provides a tractable approach, conceptually and practically, to unravel the "bird's nest" of the SCN topology. It also will allow us systematically to explain the ensemble timing functions on the basis of interacting neuronal and microcircuit properties.

Communications and Engagement
SCIENTIFIC COMMUNITY: In addition to publication, the applicant is regularly invited to speak at national and international meetings ensuring the widest possible scientific audience.
PUBLIC: MHH has extensive experience as a commentator on health-related aspects of circadian clocks, in both the general press/broadcast and in-person events (University of Cambridge BrainFest, MRC-LMB Open Days). The PI and PDRA will seek out other similar opportunities throughout the course of this proposal to maximise the public impact of this work.
PUBLIC POLICY: MHH has advised government departments on the health relevance of circadian clock and their disturbance, especially in the context of shiftwork.
THIRD SECTOR: MHH has worked with research charities e.g. AgeUK, CHDI to explore the relevance of circadian clock and their disturbance in ageing and neurodegenerative conditions.

Exploitation and application
Within the framework of the MRC-LMB/ AstraZeneca "Blue skies" research agreement MHH has direct and ready access to local Pharma groups suitable for exploitation of project findings.
Capacity and involvement
The impact activities will primarily be undertaken by the applicant with contributions from PDRA. In addition, further information will be provided via the MRC-LMB web pages. The MRC-LMB also has dedicated staff to facilitate public engagement activities, providing additional added value.

Collaboration
Collaboration is central to this proposal in the deployment of novel techniques to manipulate GABA signalling and neuronal gene expression. The GABA optogenetic control studies will use technology developed by Dr. Richard Kramer (UC Berkeley), a leading Molecular Neurobiologist. The SCN provides an ideal context in which to use it to decipher how GABAergic signalling in defined neuronal populations directs network function and thence behaviour. The amber suppression work is in collaboration with Dr. Jason Chin, MRC-LMB PNAC Division, a leading Synthetic Biologist. Whilst this approach has been developed in lower eukaryotes, our work with the Chin group has provided first-in-class experiments to reveal the applicability of the methods to mammalian brain, in cultured slices and in vivo. We anticipate that the successful progression of our programme will bring these powerful methods to the attention of a wider biomedical community.