Image-Based Profiling of Synaptic Connectivity in Primary Neuronal Cell Culture

Abstract

Neurological disorders display a broad spectrum of clinical manifestations. Yet, at the cellular level, virtually all these diseases converge into a common phenotype of dysregulated synaptic connectivity. In dementia, synapse dysfunction precedes neurodegeneration and cognitive impairment by several years, making the synapse a crucial entry point for the development of diagnostic and therapeutic strategies. Whereas high-resolution imaging and biochemical fractionations yield detailed insight into the molecular composition of the synapse, standardized assays are required to quickly gauge synaptic connectivity across large populations of cells under a variety of experimental conditions. Such screening capabilities have now become widely accessible with the advent of high-throughput, high-content microscopy. In this review, we discuss how microscopy-based approaches can be used to extract quantitative information about synaptic connectivity in primary neurons with deep coverage. We elaborate on microscopic readouts that may serve as a proxy for morphofunctional connectivity and we critically analyze their merits and limitations. Finally, we allude to the potential of alternative culture paradigms and integrative approaches to enable comprehensive profiling of synaptic connectivity.

The Fragile X Mental Retardation Protein Regulates Striatal Medium Spiny Neuron Synapse Density and Dendritic Spine Morphology

“…For example, acute expression of wtFMRP in Fmr1 KO hippocampal dissociated and slice cultures reduces total PSD95 and synapsin (unspecified) puncta, an interpretation that was bolstered by measurements of miniature excitatory postsynaptic current frequency (Pfeiffer and Huber, 2007 ). However, in our study, while we also observe reductions in PSD95 puncta counts and spine density in wtFMRP-treated cells, a similar effect is not present for colocalized puncta—a feature representative of functional synapses (Ippolito and Eroglu, 2010 ; Verstraelen et al, 2018 ). In fact, while numbers of colocalized puncta align well with overall spine density in our studies of basal conditions, when we perturb the KO cell environment with acute availability of FMRP, these outcomes no longer align, at least at the examined time point (DIV 14).…”

Identification of Potential Interacting Proteins With the Extracellular Loops of the Neuronal Glycoprotein M6a by TMT/MS

“…Axon terminals—pre-synaptic compartment—form multiple synaptic contacts with the cell soma and dendrites—post-synaptic compartment—of another neuron aided by adhesion molecules that interact across the synaptic cleft (Scheiffele, 2003 ; Sytnyk et al, 2004 ; Biederer and Scheiffele, 2007 ). These specialized junctions between neurons can be easily identified and quantified as the number of colocalization of immunoreactive synaptic clustered proteins along dendrites in cultured neurons (Dzyubenko et al, 2016 ; Verstraelen et al, 2018 ). Thus, we examined whether M6a might associate with the selected synaptic proteins by immunostaining primary hippocampal neurons at 12–15 DIV with the appropriate antibodies.…”

mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging

“…Using mGreenLantern. Spine analysis is an important and common technique in neuroscience for measuring plastic changes at brain synapses, but spine resolution is often poor with EGFP and EYFP (38). Antibody enhancement can amplify FP signal, but such treatment increases background-often introducing puncta that could be mistaken for spines (Fig.…”