Abstract
Synapse density is an essential indicator of development and functioning of the central nervous system. It is estimated indirectly through the accumulation of pre and postsynaptic proteins in tissue sections. 3D reconstruction of the electron microscopic images in serial sections is one of the most definitive means of estimating the formation of active synapses in the brain. It is tedious and highly skill-dependent. Confocal imaging of whole mounts or thick sections of the brain provides a natural alternative for rapid gross estimation of the synapse density in large areas. The optical resolution and other deep-tissue imaging aberrations limit the quantitative scope of this technique. Here we demonstrate a simple sample preparation method that could enhance the clarity of the confocal images of the neuropil regions of the ventral nerve cord of Drosophila larvae, providing a clear view of synapse distributions. We estimated the gross volume occupied by the synaptic junctions using 3D object counter plug-in of Fiji/ImageJ®. It gave us a proportional estimate of the number of synaptic junctions in the neuropil region. The method is corroborated by correlated super-resolution imaging analysis and through genetic perturbation of synaptogenesis in the larval brain. The method provides a significant improvement in the relative estimate of region-specific synapse density in the central nervous system. Also, it reduced artifacts in the super-resolution images obtained using the stimulated emission depletion microscopy technique.
