Visualising neuronal activity in cerebellar Purkinje cells

The term synapse refers to the specialised structures that allow excitable cells within the central nervous system to communicate with one another. The properties of synapses differ between cells and between parts of the brain and they can adapt over short or longer terms to modulate the strength and pattern of information transmission. Longer term changes in the strength of transmission are thought to provide a storage mechanism for learning and the process of learning may, in turn, help to sculpt patterns of information flow between networks of cells and between different structures in the brain. Understanding how synapses work, and how they can be modified, is fundamental to our understanding how the brain works and this, in turn, is an essential starting point for repairing brain function when it is damaged through injury or disease. In this proposal, we aim to generate strains of mice that have been genetically modified to express proteins that are fluorescent. These fluorescent proteins can be visualised microscopically and they alter their properties under different pH environments. By attaching these artificial proteins to natural protein structures that are involved in cell signalling and plasticity, we intend to develop methods that allow the real time visualisation of aspects of synaptic transmission, plasticity and communication in living brain cells. In the cerebellum, part of the brain necessary for the execution of skilled movement, information is transmitted from granule cells to Purkinje cells. Purkinje cells provide the sole output from this part of the brain and they are largely responsible for processing the information that enters the cerebellum. Activity within Purkinje cells triggers substantial increases in intracellular calcium, a chemical essential for cell signalling and plasticity. Calcium increases are accompanied by an acidification of the cell. By incorporating a fluorescent protein based pH sensor selectively into Purkinje cells, we aim to generate mice in which the activity of Purkinje cells can be directly visualised. We will then use brain slices prepared from these mice to evaluate how different patterns of neuronal input to the cerebellum are processed and passed on within this model network. Communication at a synapse requires the release of a chemical transmitter that diffuses across the synaptic space between the two cells and acts on a receptor present in the post-synaptic membrane to produce a response. Long-term changes in the strength of signalling between cells are thought to arise, in many cases, by either an increase or a decrease in the number of receptors present in the post-synaptic membrane. The movement of a receptor from the synaptic cleft to the inside of the cell (down-regulation) or vice verse (up-regulation), is accompanied by a sharp change in pH from the alkaline extracellular surface to the acidic inside of a transport vesicle. By tagging specific receptors expressed by Purkinje cells with a fluorescent protein pH sensor, we aim to develop mice in which we can directly visualise the movement of receptors to and from the membrane under conditions thought to produce learning. Brain slices derived from these mice will be used to examine the input conditions that produce changes in the number of receptors at a synapse and hence the strength of synaptic transmission within this part of the central nervous system. These mice will provide valuable tools to the research community and if successful, provide proof of concept for the development of other probes with uses in other parts of the brain.

The ability to learn skilled movements is thought to involve a selective long-term decrease in the strength of signalling at synapses formed between granule cells and Purkinje cells, the principal cells of the cerebellar cortex. Long-term depression (LTD) may be expressed as a down regulation of AMPA receptors which mediate fast excitatory transmission at this synapse. We have recently discovered that the properties of synapses formed by different regions of the same granule cell axon (the ascending axon segment and the parallel fibre) have very different transmission properties and are differently susceptible to forms of long-term plasticity. In this proposal, we intend to pursue why and how synapses formed by different segments of the same axon can have such fundamentally different properties and how these properties affect the transmission and plasticity of physiologically relevant information within the cerebellar cortex. To do this we will generate transgenic mice that express, selectively in Purkinje cells, one of two novel fluorescent proteins. YFpH, is a tandem, fluorescent-protein pH sensor, that has pH-sensitive and /insensitive components, allowing ratiometric quantification of intracellular pH levels. Because changes in intracellular calcium are accompanied by a reduction in intracellular pH, this sensor can be used to assess neuronal activity in the dendrites of Purkinje cells where the pH change is greatest. We have also attached this sensor to the extracellular N-terminus of the GluR2 AMPA receptors subunit. This construct allows us to estimate the relative proportions of surface expressed AMPA receptors to those inside endocytic vesicles because of the pH difference between these two compartments. These transgenic animal models will be used to examine signal processing within the cerebellar cortex and the contributions of AMPA receptor trafficking to the various forms of plasticity that occur at granule cell-Purkinje cell synapses.