SYNPLA, a method to identify synapses displaying plasticity after learning

Doré, Kim; Pao, Yvonne; Lopez, Jose Soria; Aronson, Sage; Zhan, Huiqing; Ghosh, Sanchari; Merrill, Sabina; Zador, Anthony M.; Malinow, Roberto

doi:10.1073/pnas.1919911117

Cited by 11 publications

(11 citation statements)

References 26 publications

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“…To maximize the efficiency of the PLA reaction, we selected within our panel of antibodies those that would bind markers that have been described to directly interact. From our panel, Neurexin1b was the most suitable pan-synaptic marker antibody because it interacts at the molecular level with the extracellular epitopes of four postsynaptic proteins with available antibodies, namely, GluA1-4, GABA-R, Neuroligin1c, and Neuroligin2 ( Dore et al., 2020 ; Mondin et al., 2011 ; Sudhof, 2017 ; Zhang et al., 2010 ). Indeed, all four combinations yielded the characteristic punctate PLA pattern at the expected location: inhibitory synapse markers (GABA-R and Neuroligin2) preferentially located near neuronal somas, whereas excitatory PLA signals (GluA1-4 and Neuroligin1c) were found evenly distributed along the neurites ( Figure 4 A).…”

Section: Resultsmentioning

confidence: 99%

“…PLA had been used before to confirm synaptic interactions in the pre- or post-synaptic compartment ( Almandoz-Gil et al., 2018 ; Eagleson et al., 2013 ; Lundgren et al., 2015 ). Very recently, the utility of PLA for monitoring transsynaptic interactions was demonstrated as well ( Dore et al., 2020 ). In this work, co-cultures of neurons were used in which one population was transfected with myc-tagged Neurexin and another with hemagglutinin-tagged Neuroligin.…”

Section: Discussionmentioning

confidence: 99%

“…Remarkably, despite lesser IF performance (e.g., AFC metrics), the PLA combination for Neurexin1b-Neuroligin1 performed equally well as Homer-Bassoon. Neurexin1b was even shown to be homogeneously expressed at the dendritic surface ( Dore et al., 2020 ) reducing its SBR below a standard required for spot detection. This suggests that PLA is able to discriminate the synaptic pool by virtue of its proximity dependence.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, to improve the sensitivity and reproducibility of synapse density quantification, we implemented a transsynaptic proximity-ligation assay (PLA). Considering the unique property of PLA to only detect protein interactions at distances below 40 nm ( Fredriksson et al., 2002 ), we identified antibodies that label bona fide partners at the pre- and postsynaptic sides obviating the need for overexpression of myc-tagged variants as was recently reported ( Dore et al., 2020 ). We show that transsynaptic PLA has the specificity and sensitivity necessary to provide reliable synapse counts for in vitro experiments aimed at synaptic modulation.…”

Section: Introductionmentioning

confidence: 99%

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Systematic Quantification of Synapses in Primary Neuronal Culture

et al. 2020

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Summary Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Systematic Quantification of Synapses in Primary Neuronal Culture

et al. 2020

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“…While NMDAR antagonists certainly have revolutionized the treatment of MDD in a general population, caution should be exercised when utilizing them to treat individuals with MDD and mTORC1-related diseases, and combination therapy should be considered. Dore et al use a similar PLA assay to detect synapse changes [61].…”

Section: Discussionmentioning

confidence: 99%

Role of FMRP in rapid antidepressant effects and synapse regulation

et al. 2021

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Rapid antidepressants are novel treatments for major depressive disorder (MDD) and work by blocking N-methyl-D-aspartate receptors (NMDARs), which, in turn, activate the protein synthesis pathway regulated by mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Our recent work demonstrates that the RNA-binding protein Fragile X Mental Retardation Protein (FMRP) is downregulated in dendrites upon treatment with a rapid antidepressant. Here, we show that the behavioral effects of the rapid antidepressant Ro-25-6981 require FMRP expression, and treatment promotes differential mRNA binding to FMRP in an mTORC1-dependent manner. Further, these mRNAs are identified to regulate transsynaptic signaling. Using a novel technique, we show that synapse formation underlying the behavioral effects of Ro-25-6981 requires GABABR-mediated mTORC1 activity in WT animals. Finally, we demonstrate that in an animal model that lacks FMRP expression and has clinical relevance for Fragile X Syndrome (FXS), GABABR activity is detrimental to the effects of Ro-25-6981. These effects are rescued with the combined therapy of blocking GABABRs and NMDARs, indicating that rapid antidepressants alone may not be an effective treatment for people with comorbid FXS and MDD.

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AOP report: Development of an adverse outcome pathway for deposition of energy leading to learning and memory impairment

Sleiman,

Miller,

Flores

et al. 2024

Environ and Mol Mutagen

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Understanding radiation‐induced non‐cancer effects on the central nervous system (CNS) is essential for the risk assessment of medical (e.g., radiotherapy) and occupational (e.g., nuclear workers and astronauts) exposures. Herein, the adverse outcome pathway (AOP) approach was used to consolidate relevant studies in the area of cognitive decline for identification of research gaps, countermeasure development, and for eventual use in risk assessments. AOPs are an analytical construct describing critical events to an adverse outcome (AO) in a simplified form beginning with a molecular initiating event (MIE). An AOP was constructed utilizing mechanistic information to build empirical support for the key event relationships (KERs) between the MIE of deposition of energy to the AO of learning and memory impairment through multiple key events (KEs). The evidence for the AOP was acquired through a documented scoping review of the literature. In this AOP, the MIE is connected to the AO via six KEs: increased oxidative stress, increased deoxyribonucleic acid (DNA) strand breaks, altered stress response signaling, tissue resident cell activation, increased pro‐inflammatory mediators, and abnormal neural remodeling that encompasses atypical structural and functional alterations of neural cells and surrounding environment. Deposition of energy directly leads to oxidative stress, increased DNA strand breaks, an increase of pro‐inflammatory mediators and tissue resident cell activation. These KEs, which are themselves interconnected, can lead to abnormal neural remodeling impacting learning and memory processes. Identified knowledge gaps include improving quantitative understanding of the AOP across several KERs and additional testing of proposed modulating factors through experimental work. Broadly, it is envisioned that the outcome of these efforts could be extended to other cognitive disorders and complement ongoing work by international radiation governing bodies in their review of the system of radiological protection.

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SYNPLA, a method to identify synapses displaying plasticity after learning

Cited by 11 publications

References 26 publications

Systematic Quantification of Synapses in Primary Neuronal Culture

Systematic Quantification of Synapses in Primary Neuronal Culture

Role of FMRP in rapid antidepressant effects and synapse regulation

AOP report: Development of an adverse outcome pathway for deposition of energy leading to learning and memory impairment

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