The role of presynaptic calcium at ageing synapses

Lead Research Organisation: University of Leicester
Department Name: Cell Physiology and Pharmacology


Ageing leads to a decline in our ability to remember. Some people age relatively well whereas others are very severely affected. It was originally thought that the age-related decline in cognitive function was due to a loss of cells within the brain but it is now thought that natural ageing is distinct from neurodegenerative conditions, such as Alzheimer's disease, where cell death and structural changes are very apparent. In regions of the brain responsible for memory formation and retention, such as the prefrontal cortex and hippocampus, relatively subtle changes in the connections between cells may account for age-related deficits in memory formation. Calcium plays a fundamental role in controlling many functions within cells including communication between cells. Changes in the strength of signalling between cells are thought to provide a mechanism for memory formation and these are highly dependent up calcium. We have developed a genetically modified mouse that expresses a sensor that is capable of measuring calcium. The sensor is located at the specialised terminals (synaptic terminals) of excitable cells within the brain that are responsible for releasing the chemical transmitters that transfer information from cell to cell. In this application, we wish to use imaging techniques to measure the changes in calcium within synaptic terminals during normal activity within the hippocampus and compare them to signalling in aged animals that display an age-related decrease in memory. In so doing, we hope to understand the mechanisms that are responsible for age-related decreases in brain function.

Technical Summary

Ageing is associated with a loss of memory. Subtle changes in synaptic structure and function in the prefrontal cortex and hippocampus may account for such age-related deficits in cognitive function. Ageing is accompanied by alterations in the homeostatic mechanisms that control calcium signalling. These can have profound affects on synaptic transmission and the induction of synaptic plasticity, the process that underpins memory formation. Here we wish to examine how ageing affects calcium signalling, specifically in presynaptic terminals of the hippocampus.

We have developed a transgenic mouse that expresses a novel, genetically encoded, ratiometric calcium sensor selectively at presynaptic terminals, with particularly high expression in the hippocampus. The sensor consists of a form of GCaMP linked to mCherry, which is in turn fused to the vesicular protein synaptophsin. Driven by the thy1.2 promotor, the sensor is highly expressed in the hippocampus and precisely targeted to presynaptic terminals. The mCherry signal serves two purposes. First it identifies the presynaptic terminals where the sensor is located, and second, it can be used to quantify the calcium signal from the calcium sensor through ratiometric methods. In the CA1 region, stimulation of Schaffer collateral fibres leads to a robust calcium signal. Similar responses are observed in dentate gyrus and CA3. Sensor expression increases with development allowing measurement of presynaptic calcium from ageing animals.

We wish to use this unique mouse model to characterise the contribution of presynaptic calcium to synaptic transmission at each of the main relays of the hippocampal circuit. We will establish how the calcium signals are modified by pharmacological intervention at inhibitory and excitatory circuits and how intracellular stores influence presynaptic calcium. We will then examine whether presynaptic calcium signalling is modified in mice with age-related deficits in hippocampal function.

Planned Impact

Preliminary to this application, we received BBSRC funding to develop a series of sensors that allow aspects of synaptic activity to be measured optically in real time. One of the calcium sensors has been use to generate two strains of transgenic mouse. One of these strains provides the experimental model for this application. Sensor expression is under the control of the thy1.2 promotor. In the first mouse, expression is observed in most regions of the brain we have so far studied but it is particularly high in hippocampus. This provides a tool that is potentially very useful to the entire neuroscience community because we can use these mice to look at presynaptic activation in models ranging from dissociated cultures, to organotypic cultures to in vivo measurements in awake, behaving animals. The mice can be used to detect synaptic connectivity and measure signalling strength and, as such, should be hugely useful to the entire neuroscience community because it will be possible to examine precisely where and when presynaptic inputs to neurones and non-neuronal cells in the brain are activated and how they are modified.

We have developed a whole series of sensors that work on a quantitative principle. We have combined a fluorescent protein sensor of hydrogen or calcium ions with a spectrally distinct fluorescent protein that does not respond to changes in concentrations in either of these ions. This allows us to express the change in ion concentration (calcium or pH) with respect to the amount of protein expressed and this makes calibration possible. By fusing the sensor to proteins of interest, we can target expression specifically to presynaptic compartments to measure residual calcium or transmitter release. Quantification therefore brings opportunities for comparisons between different cell types and over time. It also lends itself to use for high throughput measurements. Drug companies may be interested, for example, in screening drugs to see how they influence transmitter release or secretion. The sensors can also be used to examine absolute changes over time and so we predict that they may be used to examine long-term changes in synaptic signalling during the progression of diseases. The transgenic mouse can also be interbred with other animals and so we envisage it will be possible to make transgenic mice that have both a model of disease such as Alzheimer's disease as well as a sensor that allows a direct measurement of potential changes in synaptic transmission during disease progression.

In order to publicise the importance of these sensors, we need to demonstrate their use in situ and this is one of the corollary aims of this application. We have now characterised the sensors in model systems and used them in hippocampal cultures to examine synaptic signalling and we will submit the work for publication in the next few weeks. Academic beneficiaries will be informed through the usual means of publication and conference attendance but we will also bring this to the attention of our enterprise office, members of which actively inform various companies about technologies developed at Leicester University.

Other beneficiaries include the named PDRA, Joanna Shaw, who will receive training in a range of disciplines including electrophysiology, imaging and molecular biology as well as generic skills such as data analysis. These methods and techniques will stand her in good staid to pursue her goal of an academic career. This work also makes use of a high-speed digital microscope that we have developed with BBSRC support. We have recently obtained follow-on-funding to further develop the microscope in consultation with Prior Scientific with the aim of producing a commercial version in the near future.
Description This grant aims to examine how ageing affects the way in which the brain maintains a normal balance of calcium. Specifically we aim to establish how calcium at the presynaptic side of the synapse controls the release of transmitter and how changes in transmitter release affect the mechanisms that give rise to memory formation and storage. To do this work, we previously developed fluorescent protein sensors (BB/C508377/1 ; BB/C508377/2) and a transgenic mouse (BB/E001246/1 ) that expresses one of these sensors. It is expressed in presynaptic terminals and enables a real-time readout of synaptic activity in young and old tissues. Since this project is still in the early stages, we are currently working on younger tissue whilst we develop a colony of ageing mice. Our results currently indicate that presynaptic calcium is affected during the ageing process. We have found that in aged mice, the absolute concentration of calcium in presynaptic terminals within the CA1 region of the hippocampus is elevated. Moreover, the extent of calcium increase during stimulation is also raised, indicating that synaptic transmission is affected. These observations show that aging is associated with altered calcium homeostasis in presynaptic terminals and suggest that the basic patterns of synaptic transmission within the hippocampus will be altered. We are now pursuing funding to extend this research so that we can establish how neuronal signalling is affected in aging mice and crucially how this process can be reversed.
Exploitation Route The transgenic mouse that we have developed will be of use to the wider neuroscience community as it is a unique resource that allows the detection and measurement of calcium in presynaptic terminals. We are hoping to publish the model soon and then we will make it available. We are also generating an ageing colony of mice. We are now advertising the colony within the university and beyond in the hope that others will be able to make use of tissues or animals so that we can follow the principles of the NC3Rs.
This work is now published and the paper was highlighted in Science Magazine as an editors pick for their readers. The work has also triggered a collaboration with the US. We have submitted a grant to use our transgenic mice to breed with mouse models of Alzheimer's disease in order to examine whether neurodegneration, as with aging, leads to a change in synaptic properties.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description This grant has now finished and the manuscript published in the top international aging journal. The work was selected for highlight by Science Magazine since the observations from this wok are potentially really important as we have discovered some important changes in calcium signalling associated with age-dependent learning impairment. As a result of this and another paper, we were contacted and asked to collaborate on a large US based grant to use our transgenic mice to examine similar effects in models of neurodegeneration.
First Year Of Impact 2019
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

Description ALERT 14
Amount £638,019 (GBP)
Funding ID BB/M012034/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 11/2015
Title A miniature microscope for brain imaging in awake, behaving animals 
Description We have developed a new, miniature microscope that can be mounted on to the head of rats or mice to allow fluorescent imaging of neurones in the brain 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact We have only just developed this and so we have shown proof of concept. We have also obtained funding from within the University of Leicester in collaboration with the Space Research Centre to adapt the microscope for infra-red imaging and for multiple wavelength imaging 
Title NiMan 
Description A suite of software that allows time resolved analysis of large stacks of images but also including a novel method for creating and analysing super-resolution images from structured illumination microscopy 
Type Of Technology Software 
Year Produced 2015 
Impact The software has been developed over many years and is used by members of my laboratory and collaborators to analyse data collected from the various research projects we have undertaken over the last 10 years 
Title NiMaq 
Description A suite of software that allows the control of imaging equipment including cameras, light sources, digital RF synthesisers, microscopes and scanning devices 
Type Of Technology Software 
Year Produced 2016 
Impact This software has been developed over many years and is the basis for most of the technical developments my lab has made over many years. We hope that it will either be released as an open source tool or form part of a spin out product that we can licence or sell 
Title RF Synthesiser External Operation (XOP) for Igor Pro 
Description A driver that allows control of a radiofrequency digital synthesiser for control of acousto-optic devices 
Type Of Technology Software 
Year Produced 2016 
Impact This is central to the development of our Super-resolution microscopes that use acousto-optics and forms the basis behind any future commercial products based upon this technolgy. It is central to the operation of our super-resolution facility that uses technology invented through BBSRC funding and which is currently used by members of our university