Functional Viability: Measurement of Synaptic Vesicle Pool Sizes

Abstract

Neurons and their function of conveying information across a chemical synapse are highly regulated systems. Impacts on their functional viability can occur independently from changes in morphology. Here we describe a method to assess the size of synaptic vesicle pools using live cell fluorescence imaging and a genetically encoded probe (pHluorin). Assessing functional parameters such as the size of synaptic vesicle pools can be a valuable addition to common assays of neuronal cell viability as they demonstrate that key cellular functions are intact.

A dopaminergic mechanism of antipsychotic drug efficacy, failure, and failure reversal: the role of the dopamine transporter

“…We investigated whether longer APD treatment regimens that mimic APD efficacy and failure would affect dopamine exocytosis. We transfected cultures of hippocampal neurons, which should exhibit similar vesicular release mechanisms to dopamine neurons [46], from juvenile rats with synapto-pHIuorin (spH) and recorded synaptic vesicle exocytosis using real-time fluorescence microscopy after 1 h, 6 days or 14 days of treatment with HAL or vehicle control in culture [47]. We measured the sizes of two distinct vesicle pools, the readily releasable pool (RRP) and the recycling pool (RP) [48].…”

Visualization of synaptic vesicle dynamics with fluorescence proteins

“…For example, pHluorin is a widely used pH-dependent variant of GFP whose fluorescence is quenched at acidic pH while recovered after being exposed to near-neutral pH in the solution [2]. As the pH of SV lumen changes from acidic to near-neutral during exocytosis and re-acidic after endocytosis, the changes in fluorescence intensity caused by varying pH can reflect SVs' recycling and can be used for quantitative assessment [50,93]. Moreover, a set of similar FPs have been attained via different directed point mutations [10,61,89].…”