The role of presynaptic calcium at ageing synapses

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.

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.

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.