Nelson, Laura; (2002) An investigation of the metabotropic glutamate receptor of hippocampal neurones with flash photolysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

L-glutamate is an excitatory amino acid acting on ionotropic receptors, which mediate fast synaptic transmission, and metabotropic glutamate receptors (mGluRs), which are thought to modify excitability by modulating potassium channels and non-selective cation channels. The aims of this project are to characterise the ionic basis of the mGluR response in hippocampal CA1 pyramidal cells, to identify channels that are responsible for the effect and to investigate underlying intracellular mechanisms. Flash photolysis of 'caged' ligands is a powerful tool for time-resolved studies of the post-synaptic effects of neurotransmitters and the intracellular effects of caged second messengers. It can be used with whole-cell patch clamp and fluorescent calcium indicators to measure ionic currents and intracellular calcium concentrations ([Ca²⁺]i). The characterisation of a novel caged L-glutamate used in these experiments is described. There are two components to the mGluR response; early, activating after about half a second, and late, activating after about a second. The two components have different reversal potentials, implying that there are two different channels, selective for different ions. There is a [Ca²⁺]i rise associated with the late mGluR response. Ion substitution and pharmacological experiments identified the late channel as potassium-selective. The early component appeared to be a voltage-sensitive non-selective cation conductance. Antagonists acting selectively at different mGluR-subtypes indicated that the late potassium channel is linked to activation of a Group I mGluR and the early channel to a Group II mGluR. In many cells. Group I mGluRs activate the inositolphosphate (IP) intracellular pathway to increase [Ca²⁺]i, which activates calcium-sensitive potassium channels. Inositol 1,4,5-trisphosphate (IP3), released by flash photolysis in pyramidal cells, resulted in a potassium current that resembled the late mGluR current in timecourse and amplitude. Thus, the photorelease of IP3 mimics the late component of the mGluR response, both in [Ca²⁺]i increase and activation of the potassium conductance, indicating that the IP pathway mediates the late inhibitory response.

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