RT Journal Article SR Electronic T1 Fluorescence-based quantitative synapse analysis for cell-type specific connectomics JF eneuro JO eNeuro FD Society for Neuroscience SP ENEURO.0193-19.2019 DO 10.1523/ENEURO.0193-19.2019 A1 Dika A. Kuljis A1 Eunsol Park A1 Cheryl A. Telmer A1 Jiseok Lee A1 Daniel S. Ackerman A1 Marcel P. Bruchez A1 Alison L. Barth YR 2019 UL http://www.eneuro.org/content/early/2019/09/23/ENEURO.0193-19.2019.abstract AB Anatomical methods for determining cell-type specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed genetically-encoded reagents for fluorescence-synapse labeling and connectivity analysis in brain tissue, using a fluorogen-activating protein (FAP)- or YFP-coupled, postsynaptically-localized neuroligin-1 targeting sequence (FAP/YFPpost). FAPpost expression did not alter mEPSC or mIPSC properties. Sparse AAV-mediated expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific detection of putative synapses across diverse neuron types in mouse somatosensory cortex. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, FAPpost puncta, and presynaptic neurites in transgenic mice with saturated labeling of parvalbumin (PV), somatostatin (SST) or vasoactive intestinal peptide (VIP)-expressing neurons using Cre-reporter driven expression of YFP. Subtype-specific inhibitory connectivity onto L2 neocortical pyramidal (Pyr) neurons was assessed using automated puncta detection and neurite apposition. Quantitative and compartment-specific comparisons show that PV inputs are the predominant source of inhibition at both the soma and the dendrites and were particularly concentrated at the primary apical dendrite. SST inputs were interleaved with PV inputs at all secondary- and higher-order dendritic branches. These fluorescence-based synapse labeling reagents can facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.Significance Statement High-throughput quantitation of synapse number and distribution can reveal principles of circuit function and their adaptive or pathological alterations. Molecular genetic, fluorescence-based approaches targeted to discrete cell types can enable automated detection and quantification of input-specific synapses in complex brain tissues. In addition, these tools present a low barrier to use within the neuroscience community through volumetric confocal analysis of tissue specimens. Here we evaluate inhibitory synapse distribution across layer 2 (L2) pyramidal neurons using postsynaptic expression of a previously characterized, neuroligin-based construct. We find that inhibitory inputs from fluorescently-labeled parvalbumin and somatostatin neurons are intermingled across the proximal dendrites, and that inputs from vasoactive-intestinal peptide neurons are rare for L2 pyramidal neurons.