WIERDA-HETEROGENEITY

Analyzing Heterogeneity in Release of Synaptic Vesicles

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 Obiettivo del progetto (Objective)

'Recent studies in the Calyx of Held, a specialized synapse in the auditory pathway, have now produced clear evidence of heterogeneity in release-‘willingness’ among releasable vesicles. The general aim of this project is to investigate heterogeneity of release willingness in typical CNS synapses and to characterize the molecular mechanisms that control this heterogeneity. We will first develop a method that allows single synapse recording in primary cultured hippocampal neurons using pharmacological isolation of single visually identified (fluorescently labelled) synapses. By combining electrophysiology, fluorescence microscopy and pharmacological tools (e.g. hyperosmotic sucrose to assess total synaptic release pool size), we will perform a detailed description of (the heterogeneity in) synaptic vesicle release from single synaptic boutons. In addition, the combined research methods permit simultaneous pre- and postsynaptic assessment of neurotransmission efficacy, allowing us to pioneer in segregating pre- and postsynaptic plasticity mechanisms. Furthermore, a recently developed EGTA-based calcium-cage (DMNPE-4) allows stringent control over presynaptic Ca2 concentrations, thereby enabling detailed analysis of Ca2-sensitivity of synaptic vesicles within a single presynaptic terminal. Similar analyses will be performed in neurons deficient for candidate molecules or expressing mutated candidate molecules, thereby illuminating their role in controlling release willingness and/or Ca2 sensitivity of synaptic vesicles. A detailed understanding of these molecular mechanisms is a fundamental starting point for unravelling synaptic mechanisms behind learning and memory. In general, these efforts will provide new insights in synaptic processing and plasticity, bring us closer to the understanding of human pathogenic mechanisms in the terminal and may provide insights into the pathology of ‘synaptopathies’.'

Introduzione (Teaser)

An in-depth understanding of molecular mechanisms is essential to start discovering how synaptic activity in the brain influences learning and memory.

Descrizione progetto (Article)

The EU-funded project 'Analysing heterogeneity in release of synaptic vesicles' (Wierda-Heterogeneity) focused its research on identifying and isolating single active synapses. Such an achievement means that future research would be able to study typical synaptic activity in the central nervous system (CNS) and how that affects the storage of information in neuronal networks.

To reach its goal, the team first had to isolate single CNS synapses by blocking general synaptic transmission in neurons taken directly from a study subject. It achieved this with an infusion of postsynaptic receptor blockers, and then by locally 'unblocking' a single identified synapse.

Using a multidisciplinary approach that brought together electrophysiology, fluorescence microscopy and pharmacological tools, the research team was able to assess the efficiency of neurotransmission both before and after synaptic firing. This also allowed Wierda-Heterogeneity to take research forward in separating pre- and postsynaptic plasticity mechanisms. Plasticity is the ability of cell networks to modify their activity as required in order to encode and retain memories. Thus, efficient plasticity mechanisms are also important in learning.

As part of the study, project partners focused on the involvement of Synaptotagmin and SNAP-25 candidate genes which are prone to regulate heterogeneity in synaptic vesicle release. In the meantime, individual synapses have been visually identified in neurons in the hippocampus. Also, analyses were performed using neurons without candidate molecules or expressing mutated candidate molecules so as to highlight their role in the efficiency of pre-synaptic release.

Wierda-Heterogeneity's research efforts promise new insights into synaptic processing and plasticity. This, in turn, will enhance understanding of human pathogenic mechanisms and offer greater understanding into the pathology of synaptopathies.