Caldwell, M.B.; (2012) Application of scanning ion conductance microscopy to localised patch clamp recording from presynaptic terminals. Doctoral thesis , UCL (University College London).

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Abstract

The spatial distribution of ion channels in different subcellular regions is a key determinant of neuronal behaviour. Patch clamp electrophysiology allows characterisation of ion channel activity, but precise localisation is more difficult. This is particularly true for very small, specialised compartments such as synaptic terminals, which are inaccessible by conventional, direct patch recording methods. Scanning ion conductance microscopy (SICM) generates high resolution topographic images by using a precisely positioned probe to measure ion currents. The development of a new ‘hopping’ mode allows convoluted neuronal networks to be imaged using the SICM probe. Some details of the implementation of this mode are described. The geometry of SICM pipette tips is examined, and the interaction between the probe and the cell membrane is shown to differ from the standard account. Application of SICM to localised patch clamp recording has previously been demonstrated in several cell types and here is extended to record from presynaptic sites. Control experiments are performed and a model is introduced to explain how the use of fine-tipped SICM pipettes may give rise to artefacts seen in some of these experiments. The technique is then applied to synaptic boutons in primary cerebellar culture and a number of successful recordings are presented, along with some attempts to combine the advantages of SICM positioning with those of more conventional patch clamp pipettes. Experimental limitations of these approaches are discussed.

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