The analogue and digital clock: Role and mechanisms of analogue signalling in the brain's master circadian clock and its outputs.

Circadian (~24h) rhythms pervade many aspects of our physiology and behaviour. For example, our body shows daily variation or rhythms in metabolism, cognition, cardiovascular output, hormonal production, arousal, and the sleep-wake cycle. Disruption of these rhythms as a result of our modern 24h society lifestyles (e.g. chronic jet-lag or shift-work) can have wide implications for some serious illnesses, such as mental health, metabolic syndrome, cardiovascular disease, sleep disorders, and cancer.

In mammals, including humans, circadian rhythms are orchestrated by a master circadian clock within the brain, called the suprachiasmatic nucleus (SCN), through the rhythmic and synchronised activity of the so-called core clock genes/proteins (the molecular clock). It was long believed that in order to coordinate circadian rhythms in our body, SCN neurons discharge a type of electrical signal called action potentials (APs: an all or nothing digital code), whose frequency increases during the day and falls at night. However, we now know that there are at least two types of SCN neurons, those that contain the molecular clock (clock neurons), and those that do not (non-clock neurons). We also know that the digital peak of firing activity during the day is, in fact, mostly the activity of non-clock neurons. Remarkably, throughout most of the day, clock neurons become so excited (hyperexcited) that they stop generating APs, and instead, they start signalling with a continuous wave, the "analogue" code. Since this analogue code is the electrical output of clock neurons, we argue here that it must be critical for the overall functioning of the SCN and circadian timekeeping. Sadly, as yet, unlike its digital counterpart, the role of analogue signalling in SCN function remains unknown.

In this proposed work, we will use state-of-the-art tools previously unavailable in neuroscience research, to investigate the importance of analogue signalling in the SCN, by examining the crucial link between the clock and non-clock neurons. We will also examine the signal that is emitted by clock neurons when they start communicating in the analogue code, and investigate how this cooperates with the non-clock digital signal to broadcast circadian timing to the brain and body.

This work will bridge a critical neurophysiological knowledge-gap in our understanding of the functional relationship between the molecular clock and SCN neuronal activity. The results will provide a step change in our knowledge of how circadian rhythms are generated and communicated in the SCN, and signalled to its brain targets. This will arm us with the necessary knowledge of how to fix a "broken clock" following disruptions, as during jet-lag, shift work, disease and ageing.

Circadian (~24h) rhythms pervade many aspects of our physiology and behaviour. For example, we show daily variation in cognition, cardiovascular output, and arousal. Disruption of these rhythms leads to poor health and/or disease. In mammals, circadian rhythms are generated and coordinated by the suprachiasmatic nucleus (SCN), through the rhythmic expression of clock genes/proteins (the molecular clock).

For over 30 years it was believed that to drive circadian rhythms, the activity of the molecular clock is translated into a circadian pattern of SCN action potential (AP - the "digital" code) firing, which peaks during the day and low frequency at night. We now know that there are at least two major populations which exhibit clear and distinct molecular and electrical properties, the SCN clock and non-clock neurons. The digital peak in firing activity during the day is, in fact, mostly the result of non-clock SCN neuronal activity. Remarkably, throughout most of the day, SCN clock neurons become hyperexcited, entering depolarization blockade where they cannot generate APs. Instead of spiking, these neurons display low-amplitude membrane oscillations, the "analogue" code.

To investigate the role that analogue signalling in the SCN plays in circadian timekeeping, we will combine the sensitivity, specificity and sophistication of whole-cell electrophysiology with large-scale multi-electrode recordings, calcium imaging, optogenetics and computational modelling. Using these methods, we will first test how the hyperexcited state affects the functional relationship between SCN clock and non-clock neurons and the overall activity of the SCN network. Then, using a known SCN brain target, we will confirm the role of SCN analogue signalling in driving circadian outputs. In summary, this work will provide a crucial link between SCN molecular clock activity and SCN neurophysiology that is required to drive circadian rhythms in physiology.

This work closely aligns with the BBSRC Strategic Priorities on animal welfare, lifelong heath, and systems and a multi-disciplinary approach to bioscience research. The answers we seek in this proposal are of major interest to ACADEMIC GROUPINGS in Biological and Medical Sciences, as well as researchers in the theoretical/modelling community. The academic community will benefit enormously with the knowledge of new mechanisms by which the master circadian clock in the brain generates and communicates circadian rhythms in our body to shape the daily cycles in our physiology and behaviour, such as our sleep-wake cycle and cardiac activity. Fundamental neurophysiology data at single-cell and circuit levels, and physiology data will be made available to our collaborators, and eventually to the whole modelling community. Understanding the basic neurophysiological mechanisms of circadian rhythm timekeeping with the view to generate greater understanding of how our modern 24-hour lifestyle affects its organization and eventually our health and welfare will be of great interest to the GENERAL PUBLIC and HEALTH CARE COMMUNITY.

Even more so, if through these answers we can provide tangible mechanisms that can be targeted and exploited through appropriate patient-centered therapies to repair or alleviate debilitating internal clocks misalignment that arises due of our societally imposed schedules, disease and/or old age, our work will attract the interest of PHARMACEUTICAL INDUSTRIES. Indeed, the pharmaceutical industry investment in circadian biology research is rapidly growing. This has been driven by the increasing realisation that dysfunction in the circadian clock timekeeping can have wide implication for some serious illnesses, such as mental illness, sleep disorders, and metabolic syndrome.

In the context of partnerships-building to enhance the impact of this work, our laboratory is currently looking to form appropriate links with the pharma companies, which we hope in the long run can form a stable platform on which to train oncoming young researchers and health professionals.
From the viewpoint of basic research, we will communicate our findings by publishing scientific papers and review articles in high impact journals. We anticipate publishing at least 3 high-quality primary research papers. We will also present our work at national and international scientific meetings. We will deliver talks to primary and secondary schools, as well as participate in public engagement and deliver public lectures.

This proposal will offer an excellent opportunity for high-level training in circadian neurophysiology, from basic mechanisms to systems physiology. This will be initially for the PDRA and technician. However, PhD students joining our group will also benefit enormously from the methods that we will establish, as well as from the knowledge generated. This will significantly augment their employability, and fulfill a crucial need in UK biosciences which is to train upcoming young researchers in systems biology methods.