3-dimensional plasticity in the cerebellar cortex

Several forms of plasticity at granule cell-Purkinje cell synapses involve second messengers that are diffusible and so capable of trans-cellular movement. We have previously shown that forms of LTD and LTP in the cerebellum do not remain synapse specific but plasticity spreads to synapses formed by the same cell over distances of several tens of microns. In view of the anatomical structure of parallel fibres, which form numerous synapses with Purkinje cells across several millimetres of cortex, and given the involvement of diffusible second messengers such as nitric oxide in both LTP and LTD, we are characterising the extent to which plasticity spreads between cells connected by common parallel fibre inputs.

Funded by the BBSRC

Visualising Synaptic Plasticity

The insertion and removal of receptors from the post-synaptic membrane provide a mechanism for long-term changes in transmission strength at central synapses. We are currently testing the hypothesis that glutamate receptor trafficking underpins forms of plasticity at granule cell-Purkinje cell synapses.

By tagging novel, pH sensitive fluorescent protein constructs to subunits of the AMPA subtype of glutamate receptor, we can distinguish surface expressed receptors from those inside the cell and detect receptor trafficking to and from the plasma membrane. The images below show surface receptors selectively highlighted in red and their intracellular movement under conditions known to trigger LTD.

Funded by the BBSRC

Visualising vesicle movement and transmitter release

Transmitters are released from vesicles in presynaptic terminals. Inside vesicles, the pH is around 6. During release, the inside of the vesicle comes into brief contact with the extracellular environment which is at pH 7.4. By tagging parts of vesicular proteins exposed to the lumen of vesicles with pH sensitive fluorescent proteins, it is possible to visualise the process of release.

Current methods utilise a sensor called synaptopHluorin, which is a construct of the pH sensor super-ecliptic pHluorin and the vesicular protein synaptobrevin (aka VAMP). This has proved very useful but synaptopHluorin is quenched at the low pH found in non-releasing vesicles making them “invisible”. It is also expressed in non -vesicular membranes causing a significant reduction in overall sensitivity. We are now creating probes that combine a novel, fluorescent protein based pH sensor with various vesicular proteins with the aim of developing a tool that can distinguish releasing vesicles from those present in the presynaptic terminal that are not actively involved in release.

Funded by the BBSRC

High speed, random access microscope development

We have recently developed software for the control of confocal microscopes with the intentions of simplifying their use in conjunction with electrophysiological recordings. We are currently embarking on a project funded by the BBSRC to develop a new type of confocal microscope that allows high speed recording from individual cells or from selected points of interest from single cells.

This microscope uses a digital micromirror which serves as both a programmable array light source and a confocal pinhole. In combination with a high speed Em CCD camera, we can obtain confocal pictures at speeds of up to 100 fps.

Funded by the BBSRC