Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Abstract

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans- synaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

Trans-synaptic molecular context of NMDA receptor nanodomains

“…Synaptic nanoclustering and trans-synaptic alignment are conserved in evolution and observed across several synapse types 40,73,74 , the detailed characteristics of which depend on cleft-resident synaptic organizing complexes 1,9,11,14 . Further, the specific complement of these proteins differs across cell types and may individualize the nano-organization even of the same proteins at different excitatory synapse types 40 , or related ones at inhibitory synapses 73 . The ability to assemble a range of nanoscale protein relationships substantially broadens the functional range of a synapse.…”

Large donor CRISPR for whole-CDS replacement of cell adhesion molecule LRRTM2

“…The 3xALFA tag was inserted after the signal peptide along with a small linker (TS) after the tag, and IRES2-Cre was added immediately after the stop codon. For lentiviral expression, the guides and donor were combined into the pFW lentiviral backbone 18 by NEB HIFI Assembly. FLEx-mTagBFP2 was generated by NEB HIFI Assembly, replacing the mCherry-KASH in Addgene #139652 (a gift from Harold MacGillavry; http://n2t.net/addgene:139652; RRID:Addgene_139652) with the mTagBFP2 gene and replacing the hSyn promoter with the CAG promoter for improved expression levels.…”

“…We used a semi-automated ImageJ macro similar to that described in Dharmasri et al 18 to detect synapses. In brief, the macro provides user-guided image cropping, followed by automated thresholding to isolate ALFA-LRRTM2 signal, then uses the puncta detection plugin SynQuant 19 to detect synapses on knock-in neurons from the PSD-95 staining.…”

“…Dissociated, mixed-sex hippocampal cultures were prepared as previously described 18 . Knock-in preparations were infected at DIV1 with 100 ul each unconcentrated Cas9, guide/donor, and marker viruses.…”

Molecular definition of distinct active zone protein machineries for Ca2+channel clustering and synaptic vesicle priming

“…1a). To determine their relative nanoscale distribution, we used DNA Exchange-PAINT [53][54][55] . Enface synapses containing both proteins were selected, and auto-correlation functions were used to assess the distributions of Munc13-1 and CaV2.1 within single synapses.…”