Presynaptic substrates in hypothalamus as pivotal regulators of feeding behaviour

Animals control calorific intake by instructing feeding behaviour (eat or not eat) based on appetite state (hunger or satiety). Understanding this regulatory mechanism at the level of the underlying brain circuits reveals how the nervous system links internal physiological signals to actions that satisfy the animal - a critical function required for survival. Moreover, detailed knowledge of feeding control mechanisms and their potential dysfunction has major societal importance given the rising incidence of eating-related disorders in modern living. A brain region called the arcuate nucleus in the hypothalamus is thought to play an important role in the correct selection of appetite-related behaviour. In this structure, neurons that signal appetite state connect directly with neurons that instruct feeding behaviour. As such, changes in information flow at these contact points, known as synapses, are likely to be critical for feeding control. However, this circuit is still poorly defined and the nature of the changes that take place in these synapses to encode appetite state and drive the correct behaviour remains unclear.

The aim of this project is to investigate these key questions, applying our extensive previous knowledge of adaptive synaptic properties defined in other brain areas to characterize the mechanisms that tune information flow at the contact sites in the arcuate nucleus. Based on our pilot data, we hypothesize that a key regulatory site is the population of small nanoscale spherical structures in synapses, called vesicles, that contain and transmit the chemical signals that underlie transmission. We propose that the number of these vesicles, their physical arrangement in the synapse and the time they take to release chemical signal and become available for re-use, are key variables that determine information transfer, acting as a memory for storing appetite state and determining behavioural selection. To test these important ideas, we will take advantage of state-of-the-art genetic technologies which allow us to fluorescently label the individual vesicles in arcuate circuits taken from the brain, and, with sensitive optical microscopy, follow them through the process of chemical signalling. Likewise, using a powerful electron microscopy method, we will directly visualize the arrangements of these vesicles, testing how changes in their organization relates to alterations in appetite state. Applying our detailed previous knowledge of control pathways in other synapse types, we will also determine the molecular mechanisms that underlie appetite-driven changes in synaptic function. A final proof-of-principle objective will use our findings from these brain circuit experiments to test how imposed adjustments in synaptic vesicle properties actually impact on animal feeding behaviour.

Collectively, this work will provide fundamental new understanding of the control mechanisms in the brain that set calorific intake in behaving animals. This topic aligns with the BBSRC's strategic research priority area 'Bioscience for Health' which includes mechanisms of metabolic regulation as a key focus. Uncovering fundamental mechanisms of feeding control will fill in key knowledge gaps for understanding how animals maintain healthy body state, and provide new insights into the mechanisms of dysfunctional control associated with eating disorders.

Synapses are key control points for implementing stable adjustments in information flow in brain circuits. To date, adaptive mechanisms of synaptic tuning have focused mainly on generic networks where behavioural consequences are obscure. Here we will characterize a form of synaptic plasticity in the arcuate nucleus, a hypothalamic brain region containing circuits that critically change their signalling properties with appetitive state (hunger/satiety) to regulate feeding behaviour (eat/not eat). At present, the fundamental mechanisms that control this bistable switch are poorly understood and will be examined here. This accessible circuit is highly-advantageous for investigation, with orexigenic and anorexigenic compounds substituting for feeding motivation and the full cellular expression of hunger state retained in brain slices. Our pilot data already implicates presynaptic inputs onto agouti-related protein (AGRP) neurons as key control points. Specifically, we hypothesize that properties of the functional vesicle pools themselves - their availability for release, kinetics of recycling, and physical organization in terminals - are critical substrates for encoding appetitive state and determining feeding selection. We will test this idea in circuits where hunger state has been set, using targeted optical reporters to assay dynamic vesicle release properties and ultrastructure methods to read out recycling pool organization. Subsequently, we will characterize the key molecular pathways that determine these pool properties. To confirm the relevance of identified substrates for appetite control, we will use powerful shRNA approaches to modulate defined control mechanisms and test the impact on whole animal food-intake. We anticipate that our findings will offer major new understanding of the regulation of a key circuit underlying motivated behaviour and reveal control substrates that have direct relevance for furthering knowledge of eating-related disorders.

Who might benefit from this research? How might they benefit from this research?

1. Academic Community. The research will reveal key principles of behaviour-controlling circuit function. It will inform neuroscientists working on similar control circuits, but will also benefit the broader academic research community where knowledge of mechanistic principles of synaptic regulation is important. Findings from this research will be published in high-profile peer-reviewed journals (eg. Nature, Science, Cell, Neuron, Nature Neuroscience, as we have done in our current research) and disseminated at international meetings. Together, these benefits will enhance the knowledge economy starting in 2-5 years, with clear relevance for worldwide academic advancement. Additionally, the research plan includes the use of new and innovative technical approaches - for example, novel mouse reporter lines and groundbreaking ultrastructural approaches for readout of synaptic function. Such methods are beneficial for driving advances in understanding in many fields of neuroscience-related research. Other potential recipients of this expertise could include other academic research institutes, pharmaceutical companies, biotechnology/imaging companies and even computer technology enterprises exploring neural digital interfaces. The impact of these developments will start over 2-4 years. The work will also deliver and train highly-skilled researchers (PDRA, technician, PhD students) with expertise in organization, analysis, oral communication, and formal scientific writing skills, relevant to many employment sectors and thus further the knowledge economy. The timecourse of this benefit will start after the end of the grant.

2. Commercial Private Sector. The research will look at critical neural control points for feeding behaviour. Pharmaceutical companies looking to develop new approaches to treat eating disorders could benefit significantly from a clearer understanding of the regulatory mechanisms and molecular substrates involved. Staras has an ongoing partnership with Janssen Pharmaceuticals, formalized by a BBSRC CASE studentship, and this link is likely to be important in paving the way for the translation of findings into future therapeutic strategies. The development of specific compounds that exploit substrates identified in this project could start soon after the grant ends (3-5 years) and perhaps over 5-10 years, benefits will emerge in the form of new compounds targeting relevant substrates.

3. Public Health Sector and Economy. Given the current challenges in our population related to healthy eating and the prevalence of eating-related disorders, understanding the fundamental mechanisms that drive appetitive behaviour is a key societal issue. Benefits might be seen through development of new therapeutic approaches (above) and treatments for disorders. This could impact on the health and well-being of the nation and quality of life. Potentially, national health benefits might have economic impact through reducing strain on health services. The development of new therapeutics goes well beyond the specific aims of this grant; perhaps over a timecourse of ~10 years such benefits may start to be realized.

4. Wider Public. Our research plan has important relevance for the public understanding of science and for communicating a message of healthy eating. We will connect with the public via engagement events such as Café Scientifique, open labs, various forms of digital media, teacher conferences, school 6th form lectures, and popular articles explaining neuroscience research. Benefits will start from the beginning of the grant.