FM-Sim: Protocol Definition, Simulation and Rate Inference for Neuroscience Assays

Synaptic vesicle recycling at the presynaptic terminal of neurons is essential for the maintenance of neurotransmission at central synapses. Among the tools used to visualise the mechanics of this process is time-series fluorescence microscopy. Fluorescent dyes such as FM143, or engineered fluorescent versions of synaptic vesicle proteins such as pHluorins, have been employed to reveal di↵erent steps of this key process [3, 7]. Predictive in silico modelling of potential experimental outcomes would be highly informative for these time consuming and expensive studies. We present FM-Sim [9], user-friendly software for defining and simulating fluorescence microscopy experimental assays, with the following features: intuitive user definition of experimental protocols; automatic conversion of protocol definitions into time series rate value changes; domain-specific simulation model of a synaptic terminal; experimental data used for model parameter value inference; automatic Bayesian inference of parameter values [1, 5] and reduction of inferred parameter set size for Bayesian inference. 1 The Synaptic Vesicle Cycle Within chemical synapses of central nervous system (CNS) neurons, neurotransmitter is released from the presynaptic terminal to propagate the neural signal to the postsynaptic terminal of the following neuron. This neurotransmitter is stored in vesicles within the presynaptic terminal. These vesicles are exocytosed in response to an incoming action potential (Figure 1). To prevent vesicle depletion, compensatory endocytosis of plasma membrane allows regeneration of these vesicles. Two forms are studied within CNS nerve terminals: – Clathrin Mediated Endocytosis (CME) [6]. Individual vesicles are reconstructed directly from the plasma membrane. Following reacidification of the vesicle contents and refilling with neurotransmitter, these vesicles rejoin the vesicle pools. 2 D. Stewart, S. Gilmore, M.A. Cousin – Activity Dependent Bulk Endocytosis (ADBE) [4] is a second endocytosis mechanism triggered by periods of high stimulation. Here, large areas of plasma membrane are endocytosed as endosomes, which are later broken down into individual vesicles for reuse. FM-Sim uses a hybrid stochastic model with delays of the vesicle cycle for simulation and inference. The model supports the behaviour of di↵erent fluorescent probes. The kinetic rates and associated time delays of state transitions are the parameters of the model. 2 Fluorescence Microscopy Imaging Time-series fluorescent microscopy is one of the tools used to study the mechanisms of the synaptic vesicle cycle. Fluorescent probes added to nerve terminals allow us to obtain time-series images of nerve terminal behaviour under stimulation. The two commonly used forms of fluorescent probes are FM dyes (such as FM1-43 and FM2-10), and engineered pH-sensitive fluorescent synaptic vesicle proteins (pHluorins). The change in fluorescence of a nerve terminal as a whole over time and under changing stimuli gives insight into internal behaviour. By using either FM dyes or pHluorins in combination with chemical inhibitors, or on various knockdown animal models, di↵erent aspects of the synaptic vesicle cycle can be isolated and studied. It is this variety of potential experiments which makes FM-Sim useful at the design phase. New experiments can be simulated based upon rate parameters obtained from prior similar experiments. Exocytosis Clathrin Mediated Endocytosis Bulk Endocytosis Readily Releasable Pool Reserve Pool