Ultrastructure-function properties of recycling vesicle pools in native central synapses

The basic function of the brain is to process information: receiving sensory input, generating appropriate responses and learning/remembering. The information is encoded in the form of small electrical signals, which are passed between specialized cells called neurons wired up together to form circuits. The way the information transfers from neuron to neuron is closely controlled but also adjustable, and these properties underlie the computational power of the brain. Currently, we only have a basic comprehension of transmission; determining how neurons regulate and vary information flow is central to understanding how the brain works and is a major goal in neuroscience research.

Most information transmission occurs at chemical synapses, specialized contact points where two neurons - a signalling and receiving neuron - come close together. The signalling neuron has a cluster of spherical structures called vesicles, each containing a chemical signal. Electrical activity triggers this neuron to mobilize its vesicles and release their chemical contents towards the part of the synapse belonging to the receiving neuron. This target structure has special receptors which respond to the chemical and bring about a change in the electrical activity of the neuron. In this way, information is moved from one neuron to the next.

Why does the process involve an intermediate chemical transmission step? This allows synapses to control and adjust the information transferred. Such a feature sets networks of neurons apart from digital circuits in computers, allowing them to adapt to changes in operational demand or even 'rewire' themselves to support learning.

How is this flexible nature of transmission achieved? This is a key issue in neuroscience. One possibility is that transmitter-containing vesicles themselves are involved. Most mammalian synapses have ~250 vesicles. Surprisingly, only a fraction of these are available to release their chemical signal. If the number of releasable vesicles could be varied at each synapse, this would offer a simple mechanism to allow adjustments in the information transferred. Alternatively, the physical positioning of vesicles in synapses - allowing them to be released more or less efficiently - could be an important factor. These ideas have been difficult to address, particularly in real brain circuits, because of the technical challenges in monitoring small synapses and the nanometre scale of the vesicles.

The objective of this grant is to explore these key ideas. We will use fluorescence imaging methods to directly visualize the dynamic properties of single synapses in rat brain tissue. Also, using a novel approach we will uniquely view individual releasable vesicles with ultrastructural resolution. We will characterize the numbers of functional vesicles in different synapses in large neuronal networks, and determine what molecular pathways and other properties of circuits set these parameters. By building 3d reconstructions of vesicle populations at a synapse, we will also investigate how releasable vesicles are arranged, and examine how these properties influence synaptic performance. We will also define the characteristics of vesicle organization in synapses driven by visual input in behaving animals, allowing us to explore synaptic properties in relation to real sensory signals. Additionally, we will test whether forms of electrical input, corresponding to activity experienced by neurons during learning, bring about changes in the properties of vesicle organization. Addressing these questions is a major step in understanding fundamental brain function. Moreover, synaptic transmission is a major target for neurological diseases, such as Alzheimer's. Vesicle populations represent obvious potential substrates that could underlie synaptic failure; in the future, characterizing the mechanisms that regulate their function could offer promising new strategies for disease-related drug therapies.

Chemical synapses underlie most information transfer in the CNS. Elucidating the factors that determine their variable transmission strength and operational flexibility, endowing the nervous system with powerful computational capabilities, is a key issue in neuroscience.

The neurotransmitter-containing vesicles in the presynaptic terminal are a defining feature of synaptic architecture. Vesicles appear equivalent but only a limited subset participate in activity-driven signalling. As such, the properties of these recycling sub-pools - their size, nanoscale organization and recycling kinetics - are likely to be important determinants of synaptic transmission. They are also potential targets for plasticity, contributing to observed changes in transmission strength. However, in native central synapses, functional vesicle pools are almost completely undefined and these key ideas remain unexplored.

The proposed research will address this knowledge gap, exploiting novel methods to uniquely characterize the relationship between nanoscale and functional properties of recycling vesicles. Specifically, we will test the hypothesis that pool size and spatial organization are key determinants of presynaptic transmission under regulatory control. What specifies these parameters across synaptic populations and how substrates regulating pool organization influence synaptic efficacy will be examined. In vivo experiments in visual cortex will explore recycling pool organization related to activity driven by natural sensory input. We will also examine changes in functional pools using plasticity models (long-term potentiation or long-term depression) to test whether reorganization of functional vesicle pools contributes to plasticity-evoked modulation of synaptic strength. This work will shed light on a fundamental area of contemporary neuroscience bringing us towards an ultrastructure-function-based understanding of synaptic transmission.

The research will reveal key mechanisms regulating synaptic transmission in central mammalian neurons in native tissue. The fundamental relevance of this research topic means that the findings will be of benefit to the broad academic research community where understanding the underlying principles of neuronal signalling is important; for example researchers studying synaptic operation, neural circuit function, learning and memory, sensory processing, neuropharmacology and neurological disease. Findings from this research will be published in high-profile peer-reviewed journals, disseminated at international meetings and communicated to the public via engagement events such as Café Scientifique, open labs and various forms of media. Together, these benefits will enhance the knowledge economy starting in 2-4 years, with clear relevance for worldwide academic advancement.

Additionally, the research plan includes the use of new and innovative technical approaches - for example an in vivo method for elucidating synaptic transmission properties down to single vesicle resolution. Such methods are beneficial for driving advances in understanding in many fields of neuroscience-related research. 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. Moreover, the techniques used in this proposal are cutting-edge; 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 timecourse of this benefit will start after the end of the grant.

The research could also have societal impact. An expected outcome is the identification of new target substrates that help determine synaptic strength. These could be highly relevant to understanding forms of disease-related synaptic dysfunction - for example in Alzheimer's and Parkinson's disease - which have such catastrophic consequences for cognition and adversely affect society. Pharmaceutical companies looking to develop new approaches to treat such disorders will benefit from a clearer understanding of the regulatory mechanisms and molecular substrates that determine transmission characteristics. Ultimately, success in the development of new treatments would substantially benefit the broader public, impacting on the economy and wealth and health of the nation. Of course, 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.