Transmission at glutamate synapses in the cerebellar cortex: biophysical, pharmacological and photochemical approaches.

In the cerebellar cortex Purkinje cells receive about 200,000 excitatory parallel fibre (PF) synapses, and a single climbing fibre connexion. The parallel fibre synapses have ionotropic and metabotropic glutamate receptors associated with the postsynaptic density, the former mediating fast transmission, the latter, mainly mGluR1α, initiating intracellular signaling. We are investigating the properties, mechanisms and interactions of glutamate receptor signaling at PF synapses with electrophysiological, imaging and photochemical approaches. More generally, to study synapses and receptors in situ we are developing new probes and methods for photolytic release of amino acid neurotransmitters and other receptor ligands.
mGluR1 receptors in Purkinje cells are present at PF synapses and are needed for normal cerebellar motor coordination. This forms a background to current projects to investigate the signaling pathways and interactions with fast synaptic transmission gated directly by L-glutamate. Previously we have characterised two signaling pathways initiated at mGluR1 receptors by glutamate released from parallel fibres. One acts to release Ca2+ from internal stores in spines, activating in turn Ca2+ dependent K+ channels and is dependent on prior activity in the Purkinje cell. The second is a slow excitatory postsynaptic potential (EPSP), producing sustained excitation by prolonged depolarisation over a few hundred ms and a slow Ca2+ influx. Both pathways are G-protein coupled to mGluR1α and regulated by protein tyrosine kinase/phosphatase. Recently we have found that activation of the synaptic AMPA receptors inhibits the slow mGluR EPSC, an action mediated by a src-family protein tyrosine kinase. As a result PF synapses mediating AMPA-receptor fast EPSCs will have reduced mGluR1 activation, whereas ’silent synapses’, with no AMPAR activation, will have up-regulated mGluR1 mediated Ca2+ signaling, responding to low glutamate concentrations released at neighboring synapses.
Identification, interactions and role of postsynaptic glutamate receptors at PF synapses.
Purkinje cells express several types of ionotropic glutamate receptors (AMPA, Kainate, NMDA), comprising different combinations of receptor subunits, and also auxilliary receptor associated proteins (TARPs γ2 and γ7), and mGluR receptors. Yet PF-PC synapses are generally viewed as a uniform population with homogeneous postsynaptic properties. Current experiments aim to distinguish the contributions of the several AMPA receptor subtypes differentially represented among PF synapses during PF activation. Experiments based on differences in pharmacological and biophysical characteristics, and kinetics of calcium concentration changes, aim to identify their roles, their distribution and interactions with mGluR1 signaling in PCs.
Metabotropic glutamate receptors type 1 are important for Ca signalling and excitability, coupled to Ca release from stores in spines, to the Ca influx during the slow EPSP, to T-type Ca channesl and to an ERG K channel controlling excitability. We are studying the distribution of Ca signalling in the dendritic tree of Purkinje neurons initiated by mGluR1 activation, the interactions among the 4 downstream signaling pathways and the influence of ongoing electrical activity.