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Abstract
Presynaptic assembly involves the specialization of a patch of axonal membrane into a complex structure that supports synaptic vesicle exocytosis and neurotransmitter release. In mammalian neurons, presynaptic assembly is widely studied in a co-culture assay, where a synaptogenic cue expressed at the surface of a heterologous cell induces presynaptic differentiation in a contacting axon. This assay has led to the discovery of numerous synaptogenic proteins, but has not been used to probe neuronal mechanisms regulating presynaptic induction. The identification of regulatory pathways that fine-tune presynaptic assembly is hindered by the lack of adequate tools to quantitatively image this process. Here, we introduce an image-processing algorithm that identifies presynaptic clusters in mammalian co-cultures and extracts a range of synapse-specific parameters. Using this software, we assessed the intrinsic variability of this synaptic induction assay and probed the effect of eight neuronal microRNAs on presynaptic assembly. Our analysis revealed a novel role for miR-27b in augmenting the density of presynaptic clusters. Our software is applicable to a wide range of synaptic induction protocols (including spontaneous synaptogenesis observed in neuron cultures) and is a valuable tool to determine the subtle impact of disease-associated genes on presynaptic assembly.
Highlights
Chemical synapses are specialized cellular junctions that permit information flow from one neuron to another
Hippocampal neurons were cultured with an astroglial feeder layer to accelerate neuronal differentiation (Kaech and Banker, 2006) and transduced with a GFP-expressing lentivirus to visualize neuronal processes (Figure 1)
Neurons were fixed at DIV8, stained with an antibody against the synaptic vesicle (SV) protein synaptobrevin and imaged by confocal microscopy (Supplemental Figure 1)
Summary
Chemical synapses are specialized cellular junctions that permit information flow from one neuron to another. They consist of pre- and post-synaptic compartments, whose function is to release and respond to neurotransmitters, respectively. An action potential triggers the entry of calcium ions into the presynapse, which induces synaptic vesicle (SV) fusion and release of neurotransmitters into the synaptic cleft. This complex multi-step process is highly regulated and involves a large number of molecular components. SV components and molecules required for SV exocytosis, endocytosis, and recycling have been implicated in Alzheimer’s disease, schizophrenia, Parkinson’s disease, and other disorders (Waites and Garner, 2011)
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