High-content imaging of presynaptic assembly

Poon, Vivian Y.; Goh, Chiatzun; Voorhoeve, P. Mathijs; Fivaz, Marc

doi:10.3389/fncel.2014.00066

Cited by 9 publications

(12 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…E18 Sprague-Dawley rat pups are decapitated and utilizing aseptic technique, cortical tissue is dissected from the embryonic brain and placed directly into a 15 ml sterile plastic vial containing 10 ml ice-cold HBSS or Hibernate-E medium (BrainBits) 1 and brought to a laminar flow hood for extraction of neurons from the cortical tissue. E1 is defined as the day after the plug is determined to be sperm-positive (Poon et al, 2014 ). All procedures carried out were approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore.…”

Section: Methodsmentioning

confidence: 99%

Stimulus information stored in lasting active and hidden network states is destroyed by network bursts

Dranias

Westover

Cash

et al. 2015

Front. Integr. Neurosci.

View full text Add to dashboard Cite

In both humans and animals brief synchronizing bursts of epileptiform activity known as interictal epileptiform discharges (IEDs) can, even in the absence of overt seizures, cause transient cognitive impairments (TCI) that include problems with perception or short-term memory. While no evidence from single units is available, it has been assumed that IEDs destroy information represented in neuronal networks. Cultured neuronal networks are a model for generic cortical microcircuits, and their spontaneous activity is characterized by the presence of synchronized network bursts (SNBs), which share a number of properties with IEDs, including the high degree of synchronization and their spontaneous occurrence in the absence of an external stimulus. As a model approach to understanding the processes underlying IEDs, optogenetic stimulation and multielectrode array (MEA) recordings of cultured neuronal networks were used to study whether stimulus information represented in these networks survives SNBs. When such networks are optically stimulated they encode and maintain stimulus information for as long as one second. Experiments involved recording the network response to a single stimulus and trials where two different stimuli were presented sequentially, akin to a paired pulse trial. We broke the sequential stimulus trials into encoding, delay and readout phases and found that regardless of which phase the SNB occurs, stimulus-specific information was impaired. SNBs were observed to increase the mean network firing rate, but this did not translate monotonically into increases in network entropy. It was found that the more excitable a network, the more stereotyped its response was during a network burst. These measurements speak to whether SNBs are capable of transmitting information in addition to blocking it. These results are consistent with previous reports and provide baseline predictions concerning the neural mechanisms by which IEDs might cause TCI.

show abstract

Section: Methodsmentioning

confidence: 99%

Stimulus information stored in lasting active and hidden network states is destroyed by network bursts

Dranias

Westover

Cash

et al. 2015

Front. Integr. Neurosci.

View full text Add to dashboard Cite

show abstract

“…We have overcome these challenges through careful and systematic optimization of methods. In fact, the majority of the previous studies using HCS approaches involve immunocytochemistry-based endpoint assays that require fixation of cultured cells, which fundamentally limits comparison of changes before and after treatment and has a relative high intra-well variabilities due the uneven distribution of neurites and variabilities introduced by fixation and immunostaining 8,[30][31][32][33][34] . Microelectrode array-based assays require specific instruments and techniques and are extremely expensive for screening approaches 35 .…”

Section: Discussionmentioning

confidence: 99%

Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates

Jiang

Esparza

Zhong

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer's disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the Mtt assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications. Synapses are asymmetric intercellular junctions that permit neurons to transmit electrical or chemical signals to other neurons. Dysregulated synaptic function, abnormal synaptic plasticity and synaptic loss are involved in many neurodevelopmental and neurodegenerative disorders, in particular Alzheimer's disease (AD) 1-3. As the most common dementing disorder in the elderly, AD is tightly correlated with synaptic loss in vulnerable brain regions, which has led to the hypothesis that loss of synaptic terminals is a key event in early cognitive decline 4. Accurate quantification of mammalian central nervous system synaptic changes is important for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and developmental or pathological synapse elimination. As many neurodegenerative conditions are known to affect synapses, quantification of synapses is also critical for identifying synaptotoxic factors and for screening of potential therapeutic agents. However, few efficient methods are available for this purpose. Electrophysiology, electron microscopy, and most light microscopy approaches are costly, time-consuming, and labor-intensive. Unbiased, high throughput screening for synaptotoxic substances in brain disorders such as AD would provide a better understanding of the disease process and an opportunity to identifying new therapeutic targets and drug candidates. High content screening (HCS) combines the efficiency of high-throughput, multi-well plate-based techniques with quantitative image analysis at subcellular resolution 5-7. Recent advances in HCS have made high-throughput analysis of synaptic changes in live neurons po...

show abstract

“…Both constructs were made in a pFUGW backbone vector. Lentiviral particles based on the pFUGW vector were generated as previously described [ 24 ]. A multiplicity of infection (MOI) between two and five was used for all viral transduction experiments.…”

Section: Methodsmentioning

confidence: 99%

“…RNA was also extracted from the same three neuronal preparations for qPCR analysis using Sepasol RNA I Super G. For microarray analysis, RNA was isolated from DIV14 neurons using the RNeasy Mini kit (Qiagen) and RNA quality was analysed on a Bioanalyzer 2100 (Agilent Technologies). Rat cortical and hippocampal neurons were isolated from E18 rat embryos as previously described [ 24 ]. Lentiviruses were added 3 h or a day after plating.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

miR-27b shapes the presynaptic transcriptome and influences neurotransmission by silencing the polycomb group protein Bmi1

Poon

et al. 2016

BMC Genomics

Self Cite

View full text Add to dashboard Cite

BackgroundMicroRNAs (miRNAs) are short non-coding RNAs that are emerging as important post-transcriptional regulators of neuronal and synaptic development. The precise impact of miRNAs on presynaptic function and neurotransmission remains, however, poorly understood.ResultsHere, we identify miR-27b—an abundant neuronal miRNA implicated in neurological disorders—as a global regulator of the presynaptic transcriptome. miR-27b influences the expression of three quarters of genes associated with presynaptic function in cortical neurons. Contrary to expectation, a large majority of these genes are up-regulated by miR-27b. This stimulatory effect is mediated by miR-27b-directed silencing of several transcriptional repressors that cooperate to suppress the presynaptic transcriptome. The strongest repressive activity appears to be mediated by Bmi1, a component of the polycomb repressive complex implicated in self-renewal of neural stem cells. miR-27b knockdown leads to reduced synaptogenesis and to a marked decrease in neural network activity, which is fully restored by RNAi-mediated silencing of Bmi1.ConclusionsWe conclude that silencing of Bmi1 by miR-27b relieves repression of the presynaptic transcriptome and supports neurotransmission in cortical networks. These results expand the repressive activity of Bmi1 to genes involved in synaptic function and identify a unique post-transcriptional circuitry that stimulates expression of synaptic genes and promotes synapse differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3139-7) contains supplementary material, which is available to authorized users.

show abstract

High-content imaging of presynaptic assembly

Cited by 9 publications

References 45 publications

Stimulus information stored in lasting active and hidden network states is destroyed by network bursts

Stimulus information stored in lasting active and hidden network states is destroyed by network bursts

Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates

miR-27b shapes the presynaptic transcriptome and influences neurotransmission by silencing the polycomb group protein Bmi1

Contact Info

Product

Resources

About