Quantitative synapse analysis for cell-type specific connectomics

Abstract

Anatomical methods for determining cell-type specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed new 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). Sparse viral expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific localization of synapses across diverse neuron types in mouse somatosensory cortex. High-resolution confocal image stacks of virally-transduced neurons were used for 3D reconstructions of postsynaptic cells and automated detection of synaptic puncta. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, synapses, and presynaptic neurites to assess subtype-specific inhibitory connectivity onto L2 neocortical pyramidal (Pyr) neurons. Quantitative and compartment-specific comparisons show that PV inputs are the dominant source of inhibition at both the soma and across all dendritic branches examined and were particularly concentrated at the primary apical dendrite, a previously unrecognized compartment of L2 Pyr neurons. Our fluorescence-based synapse labeling reagents will facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.

The Impact of SST and PV Interneurons on Nonlinear Synaptic Integration in the Neocortex

“…Although SST and PV cells have relatively distinct projection patterns, the differences are more subtle than the contrast that can be achieved with local GABA iontophoresis, as used in the aforementioned work (Jadi et al, 2012). In fact, in terms of shear anatomical numbers, PV cell inputs outnumber SST cell inputs on dendrites of layer 2/3 pyramidal neurons by two-fold (Kuljis et al, 2019). However, the distribution patterns of the dendritic synapses differ for each subtype.…”

Progressive Circuit Changes during Learning and Disease

“…Finally, reciprocal synaptic connectivity between L2 and L5 pyramidal (Pyr) neurons may also provide an important source of recurrent excitation that can facilitate associative memory and integrate reward and punishment signals that occur with some delay to a sensory stimulus (Lefort et al, 2009). A ubiquitous inhibitory circuit motif found across multiple neocortical areas is composed of vasoactive intestinal peptide (VIP) inhibitory interneurons that synapse strongly onto somatostatin (SST) but weakly onto PV inhibitory neurons (Pfeffer et al, 2013) and generally avoid pyramidal neurons (Jiang et al, 2015;Kuljis et al, 2018). Consequently, the activation of VIP neurons is associated with the suppression of SST neuron activity (Lee et al, 2013), whose elevated spontaneous firing both directly and indirectly inhibits the network (Figure 2A; Urban-Ciecko and ).…”