Abstract
BackgroundIn terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis.MethodsWe have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention.ResultsIn this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone.ConclusionsOur findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.
Highlights
In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission
RIM1α levels are linearly related to the release probability (Pr) of local axon collaterals of CA3 [7], and presynaptic efficiency can be bidirectionally modulated by modifying RIM1α levels [38]
To determine the extent to which differences in the magnitude of responses between individual nerve terminals are caused by variations in protein content, we developed a method in which post-hoc immunolabelling is combined with optical tracking of the synaptic vesicle (SV) cycle with FM1-43, this is a powerful tool to study the correlation between protein content and synaptic function, in this case vesicular release, a relationship that has remained undefined for decades in studies of synaptic function
Summary
In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. A complex network of proteins is assembled at axonal sites that generate the so-called cytomatrix at the active zone (CAZ) These proteins interact with other proteins located either at the presynaptic plasma membrane or at vesicular membranes that regulate Ca2+dependent fusion of synaptic vesicles. Render the ROIset (Analyze > Analyze particles; tick the options appearing in Additional file 4: Figure S3C) and save this ROIset as a “.”.zip file; 8 Data Acquisition: Obtain the data from the different images, for which we recommend using the automatically generated background-subtracted images obtained from the IgorPro analysis in order to minimize user manipulation of the images. Immunoreactivity levels are not linearly related to the amount of protein [27], by normalizing it is possible to perform a comparative analysis between the edges of the distribution in a given population of synapses (Figure 2E)
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