WIERDA-HETEROGENEITY · Analyzing Heterogeneity in Release of Synaptic Vesicles

Analyzing heterogeneity in release of synaptic vesicles

New method allowing single synapse recording in primary cultured hippocampal neurons The general aim of this project is to further elucidate presynaptic mechanisms that control heterogeneity in release efficacy of typical CNS synapses, an essential feature for information storage in neuronal networks. By combining two essential disciplines in neuroscience (electrophysiology and fluorescence microscopy) we aimed to isolate single synapses, thereby permitting detailed analysis of basal heterogeneity in vesicular release willingness within and between individual synapses. In addition, this multidisciplinary approach permits simultaneous pre- and postsynaptic assessment of neurotransmission efficacy, allowing us to pioneer in segregating pre- and postsynaptic plasticity mechanisms in typical CNS synapses. In short, to study heterogeneity in synaptic vesicle release in typical CNS synapses it is essential to identify and isolate a single active synapse, thereby allowing characterisation of its release efficacy and vesicle content. We isolate single CNS synapses by blocking general synaptic transmission in primary cultured neurons using bath application of postsynaptic receptor blockers, while local perfusion/suction pipettes is used to 'unblock' a single identified synapse. Using neurons deficient for candidate molecules or expressing mutated candidate molecules illuminate their role in presynaptic release efficacy. In the proposed project we focused on the involvement of two candidate genes (Synaptotagmin and SNAP-25) prone to regulate heterogeneity in synaptic vesicle release. We are able to visually identify individual hippocampal synapses in primary hippocampal neurons using viral introduction of a fusion protein between vesicle-associated Synaptophysin and either a constitutive marker (mCherry) or a pH-sensitive super-ecliptic GFP (pHluorin) that allows activity-dependent changes in presynaptic GFP fluorescence. The synapse isolation method allows single synapse recording in primary cultured hippocampal neurons using pharmacological isolation of the single visually identified (fluorescently labelled) synapses. Several technical difficulties delayed imbedding this technique as a standardized tool and we are now in the process of optimising the method. Characterisation of autaptic and synaptic release in pairs of primary hippocampal neurons Recent publications question the validity of primary hippocampal self-innervating (autaptic) neurons being used as a major experimental model for studying synaptic transmission. It has been claimed that the autaptic connections that are established in this experimental model are aberrant and therefore inappropriate for studying synaptic transmission. We decided to (in parallel with the development of synapse isolation) compare synaptic and autaptic transmission using both single and paired whole cell voltage clamp recordings in primary wildtype hippocampal cultures. Heterogeneity of vesicle release efficacy within isolated hippocampal synapses Although characterising release from isolated synapses gains insights in the heterogeneity of synaptic release efficacy and its plasticity, it will not elucidate heterogeneity in vesicular release willingness within single presynaptic terminals. In other experimental preparations such heterogeneity is largely attributable to a different arrangement of vesicles and calcium channels, with 'readily-releasable' vesicles on average closer to calcium channels than 'reluctant' vesicles. Currently we are exploring the technical requirements for allowing spot focus photolysis of caged calcium surrounding a single synaptic bouton. This technique allows us to study calcium dependence of synaptic release in typical single central nervous system synapses. In addition, calcium dependence in neurons deficient for candidate molecules likely involved in regulating calcium dependent release will be studied.

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