Abstract
The morphology of synapses is of central interest in neuroscience because of the intimate relation with synaptic efficacy. Two decades of gene manipulation studies in different animal models have revealed a repertoire of molecules that contribute to synapse development. However, since such studies often assessed only one, or at best a few, morphological features at a given synapse, it remained unaddressed how different structural aspects relate to one another. Furthermore, such focused and sometimes only qualitative approaches likely left many of the more subtle players unnoticed. Here, we present the image analysis algorithm ‘Drosophila_NMJ_Morphometrics’, available as a Fiji-compatible macro, for quantitative, accurate and objective synapse morphometry of the Drosophila larval neuromuscular junction (NMJ), a well-established glutamatergic model synapse. We developed this methodology for semi-automated multiparametric analyses of NMJ terminals immunolabeled for the commonly used markers Dlg1 and Brp and showed that it also works for Hrp, Csp and Syt. We demonstrate that gender, genetic background and identity of abdominal body segment consistently and significantly contribute to variability in our data, suggesting that controlling for these parameters is important to minimize variability in quantitative analyses. Correlation and principal component analyses (PCA) were performed to investigate which morphometric parameters are inter-dependent and which ones are regulated rather independently. Based on nine acquired parameters, we identified five morphometric groups: NMJ size, geometry, muscle size, number of NMJ islands and number of active zones. Based on our finding that the parameters of the first two principal components hardly correlated with each other, we suggest that different molecular processes underlie these two morphometric groups. Our study sets the stage for systems morphometry approaches at the well-studied Drosophila NMJ.
Highlights
Normal brain function relies on functional neuronal networks in which neurons connect and communicate with one another
Surveying a large neuromuscular junction (NMJ) image repository, we provide insights in the natural variation of NMJ morphology as a result of differences in gender, genetic background and abdominal body segment
We propose that two of them, NMJ size and geometry, are controlled by different molecular mechanisms
Summary
Normal brain function relies on functional neuronal networks in which neurons connect and communicate with one another. Communication primarily takes place at chemical synapses, where neurotransmitters are released from the presynaptic compartment of a neuron and activate receptors at the postsynaptic compartment of its target cell. The morphology and function of synapses are highly intertwined [6,7,8,9] and morphological aspects have been studied extensively to gain further insight into the regulatory networks underlying synaptic function. Mammalian dendritic spines change shape upon maturation and plasticity from long, thin filopodia-like structures to typical stubby and mushroom-shaped postsynaptic compartments of increased efficacy [10,11]. In Drosophila, synaptic structure and activity is modulated according to circadian timing [12,13,14,15] or upon experienced-dependent or stimulated activity [16,17], to name only three examples
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