Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates

AbstractLong-lasting forms of synaptic plasticity such as synaptic scaling are critically dependent on transcription. Activity-dependent transcriptional dynamics in neurons, however, have not been fully characterized, because most previous efforts relied on measurement of steady-state mRNAs. Here, we profiled transcriptional dynamics of primary neuronal cultures undergoing network activity shifts using nascent RNA sequencing. We found pervasive transcriptional changes, in which ~45% of expressed genes respond to network activity shifts. Notably, the majority of these genes respond to increases or decreases of network activity uniquely, rather than reciprocally. We further linked the chromatin regulator Retinoic acid induced 1 (RAI1), the Smith-Magenis Syndrome gene, to the specific transcriptional program driven by reduced network activity. Finally, we show that RAI1 is essential for homeostatic synaptic upscaling but not downscaling. These results demonstrate the utility of bona fide transcription profiling to discover mechanisms of activity-dependent chromatin remodeling that underlie normal and pathological synaptic plasticity.

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Quantitation of Chronic and Acute Treatment Effects on Neuronal Network Activity Using Image and Signal Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113486518 ◽

2013 ◽

Vol 18 (7) ◽

pp. 807-819 ◽

Cited By ~ 11

Author(s):

Frans Cornelissen ◽

Peter Verstraelen ◽

Tobias Verbeke ◽

Isabel Pintelon ◽

Jean-Pierre Timmermans ◽

...

Keyword(s):

Neuronal Cell ◽

Network Activity ◽

Cell Segmentation ◽

Neuronal Cultures ◽

Nuclear Staining ◽

Primary Neuronal Cultures ◽

Independent Manner ◽

Neuronal Network Activity ◽

Decay Times

Upon maturation, primary neuronal cultures form an interconnected network based on neurite outgrowth and synaptogenesis in which spontaneous electrical activity arises. Measurement of network activity allows quantification of neuronal health and maturation. A fluorescent indicator was used to monitor secondary calcium influxes after the occurrence of action potentials, allowing us to examine activity of hippocampal cultures via confocal live cell imaging. Subsequently, nuclear staining with DAPI allows accurate cell segmentation. To analyze the calcium recording in a robust, observer-independent manner, we implemented an automated image- and signal-processing algorithm and validated it against a visual, interactive procedure. Both methods yielded similar results on the emergence of synchronized activity and allowed robust quantitative measurement of acute and chronic modulation of drugs on network activity. Both the number of days in vitro (DIV) and neutralization of nerve growth factor (NGF) have a significant effect on synchronous burst frequency and correlation. Acute effects are demonstrated using 5-HT (serotonin) and ethylene glycol tetra-acetic acid. Automated analysis allowed measuring additional features, such as peak decay times and bursting frequency of individual neurons. Based on neuronal cell cultures in 96-well plates and accurate calcium recordings, the analysis method allows development of an integrated high-content screening assay. Because molecular biological techniques can be applied to assess the influence of genes on network activity, it is applicable for neurotoxicity or neurotrophics screening as well as development of in vitro disease models via, for example, pharmacologic manipulation or RNAi.

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Simulation of Spontaneous Activity in Neuronal Cultures with Long-Term Plasticity

Математическая биология и биоинформатика ◽

10.17537/2015.10.234 ◽

2015 ◽

Vol 10 (1) ◽

pp. 234-244 ◽

Cited By ~ 2

Author(s):

А.А. Дегтерев ◽

A.A. Degterev

Keyword(s):

Spontaneous Activity ◽

Network Models ◽

Network Activity ◽

Neuronal Cultures ◽

Pacemaker Neurons ◽

Short Term Plasticity ◽

Synaptic Noise ◽

Stdp Rule

Existence of spontaneous population bursts is a widely studied phenomenon observed in neuronal cultures in vitro. Recent models of neuronal cultures network activity consist of a number of burst generating mechanisms such as synaptic noise and presence of pacemaker neurons in the network. In the previous simulations of bursting in neuronal cultures synaptic weights change in accordance with the rule of short-term plasticity whereas the long-term values of them, and hence the network structure, remain unchanged. In this paper we reproduce neuronal network models with static synapses, and then investigate spontaneous activity changes in neuronal networks with long-term plasticity defined by STDP rule. Our results demonstrate that introduction of long-term plasticity in the model leads to discrepancy with the experimental data.

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Difluoroboron β-diketonate polylactic acid oxygen nanosensors for intracellular neuronal imaging

Scientific Reports ◽

10.1038/s41598-020-80172-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Meng Zhuang ◽

Suchitra Joshi ◽

Huayu Sun ◽

Tamal Batabyal ◽

Cassandra L. Fraser ◽

...

Keyword(s):

Neuronal Activity ◽

Oxygen Sensing ◽

Brain Slices ◽

Neuronal Cell ◽

Quantum Yields ◽

Poly Lactic Acid ◽

Neuronal Cultures ◽

Primary Neuronal Cultures ◽

Non Invasive ◽

Mitotracker Red

AbstractCritical for metabolism, oxygen plays an essential role in maintaining the structure and function of neurons. Oxygen sensing is important in common neurological disorders such as strokes, seizures, or neonatal hypoxic–ischemic injuries, which result from an imbalance between metabolic demand and oxygen supply. Phosphorescence quenching by oxygen provides a non-invasive optical method to measure oxygen levels within cells and tissues. Difluoroboron β-diketonates are a family of luminophores with high quantum yields and tunable fluorescence and phosphorescence when embedded in certain rigid matrices such as poly (lactic acid) (PLA). Boron nanoparticles (BNPs) can be fabricated from dye-PLA materials for oxygen mapping in a variety of biological milieu. These dual-emissive nanoparticles have oxygen-insensitive fluorescence, oxygen-sensitive phosphorescence, and rigid matrix all in one, enabling real-time ratiometric oxygen sensing at micron-level spatial and millisecond-level temporal resolution. In this study, BNPs are applied in mouse brain slices to investigate oxygen distributions and neuronal activity. The optical properties and physical stability of BNPs in a biologically relevant buffer were stable. Primary neuronal cultures were labeled by BNPs and the mitochondria membrane probe MitoTracker Red FM. BNPs were taken up by neuronal cell bodies, at dendrites, and at synapses, and the localization of BNPs was consistent with that of MitoTracker Red FM. The brain slices were stained with the BNPs, and the BNPs did not significantly affect the electrophysiological properties of neurons. Oxygen maps were generated in living brain slices where oxygen is found to be mostly consumed by mitochondria near synapses. Finally, the BNPs exhibited excellent response when the conditions varied from normoxic to hypoxic and when the neuronal activity was increased by increasing K+ concentration. This work demonstrates the capability of BNPs as a non-invasive tool in oxygen sensing and could provide fundamental insight into neuronal mechanisms and excitability research.

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Aη-α and Aη-β peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo

Alzheimer s Research & Therapy ◽

10.1186/s13195-021-00860-1 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Maria Mensch ◽

Jade Dunot ◽

Sandy M. Yishan ◽

Samuel S. Harris ◽

Aline Blistein ◽

...

Keyword(s):

Neuronal Activity ◽

Ex Vivo ◽

Long Term Potentiation ◽

Network Activity ◽

Strongly Correlated ◽

App Processing ◽

Term Potentiation ◽

Hippocampal Network

Abstract Background Amyloid precursor protein (APP) processing is central to Alzheimer’s disease (AD) etiology. As early cognitive alterations in AD are strongly correlated to abnormal information processing due to increasing synaptic impairment, it is crucial to characterize how peptides generated through APP cleavage modulate synapse function. We previously described a novel APP processing pathway producing η-secretase-derived peptides (Aη) and revealed that Aη–α, the longest form of Aη produced by η-secretase and α-secretase cleavage, impaired hippocampal long-term potentiation (LTP) ex vivo and neuronal activity in vivo. Methods With the intention of going beyond this initial observation, we performed a comprehensive analysis to further characterize the effects of both Aη-α and the shorter Aη-β peptide on hippocampus function using ex vivo field electrophysiology, in vivo multiphoton calcium imaging, and in vivo electrophysiology. Results We demonstrate that both synthetic peptides acutely impair LTP at low nanomolar concentrations ex vivo and reveal the N-terminus to be a primary site of activity. We further show that Aη-β, like Aη–α, inhibits neuronal activity in vivo and provide confirmation of LTP impairment by Aη–α in vivo. Conclusions These results provide novel insights into the functional role of the recently discovered η-secretase-derived products and suggest that Aη peptides represent important, pathophysiologically relevant, modulators of hippocampal network activity, with profound implications for APP-targeting therapeutic strategies in AD.

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Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1997.68041590.x ◽

2002 ◽

Vol 68 (4) ◽

pp. 1590-1597 ◽

Cited By ~ 30

Author(s):

Daniel R. Davis ◽

Brian H. Anderton ◽

Jean-Pierre Brion ◽

C. Hugh Reynolds ◽

Diane P. Hanger

Keyword(s):

Oxidative Stress ◽

Rat Brain ◽

Neuronal Cultures ◽

Primary Neuronal Cultures

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Ketamine decreases neuronally released glutamate via retrograde stimulation of presynaptic adenosine A1 receptors

Molecular Psychiatry ◽

10.1038/s41380-021-01246-3 ◽

2021 ◽

Author(s):

Vesna Lazarevic ◽

Yunting Yang ◽

Ivana Flais ◽

Per Svenningsson

Keyword(s):

Cortical Neurons ◽

Intracellular Signaling ◽

Ex Vivo ◽

Forced Swim Test ◽

Glutamate Release ◽

Neuronal Cultures ◽

Extracellular Glutamate ◽

Primary Neuronal Cultures ◽

Adenosine A1 Receptors ◽

Adenosine A1

AbstractKetamine produces a rapid antidepressant response in patients with major depressive disorder (MDD), but the underlying mechanisms appear multifaceted. One hypothesis, proposes that by antagonizing NMDA receptors on GABAergic interneurons, ketamine disinhibits afferens to glutamatergic principal neurons and increases extracellular glutamate levels. However, ketamine seems also to reduce rapid glutamate release at some synapses. Therefore, clinical studies in MDD patients have stressed the need to identify mechanisms whereby ketamine decreases presynaptic activity and glutamate release. In the present study, the effect of ketamine and its antidepressant metabolite, (2R,6R)-HNK, on neuronally derived glutamate release was examined in rodents. We used FAST methodology to measure depolarization-evoked extracellular glutamate levels in vivo in freely moving or anesthetized animals, synaptosomes to detect synaptic recycling ex vivo and primary cortical neurons to perform functional imaging and to examine intracellular signaling in vitro. In all these versatile approaches, ketamine and (2R,6R)-HNK reduced glutamate release in a manner which could be blocked by AMPA receptor antagonism. Antagonism of adenosine A1 receptors, which are almost exclusively expressed at nerve terminals, also counteracted ketamine’s effect on glutamate release and presynaptic activity. Signal transduction studies in primary neuronal cultures demonstrated that ketamine reduced P-T286-CamKII and P-S9-Synapsin, which correlated with decreased synaptic vesicle recycling. Moreover, systemic administration of A1R antagonist counteracted the antidepressant-like actions of ketamine and (2R,6R)-HNK in the forced swim test. To conclude, by studying neuronally released glutamate, we identified a novel retrograde adenosinergic feedback mechanism that mediate inhibitory actions of ketamine on glutamate release that may contribute to its rapid antidepressant action.

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