scholarly journals Systematic quantification of synapses in primary neuronal culture

2020 ◽  
Author(s):  
Peter Verstraelen ◽  
Gerardo Garcia ◽  
Marlies Verschuuren ◽  
Bob Asselbergh ◽  
Rony Nuydens ◽  
...  
Keyword(s):  
Synapse Formation ◽  
Dynamic Range ◽  
Cortical Cell ◽  
Neuronal Culture ◽  
Maturity Stage ◽  
Primary Neuronal Culture ◽  
Synaptic Markers ◽  
Synapse Density ◽  
Cortical Cell Cultures

AbstractA vast set of neurological disorders is associated with impaired synaptic connectivity. Therefore, modulation of synapse formation could have therapeutic relevance. However, the high density and small size of synapses make their quantification a challenging task. To improve the reliability of synapse-oriented drug screens, we evaluated a panel of synapse-targeting antibodies for their labeling specificity on hippocampal and cortical cell cultures using quantitative immunofluorescence microscopy. For those antibodies that passed multiparametric validation, we assessed pairwise colocalization, an often-used readout for established synapses. We found that even when two pan-synaptic markers were used, the overlap was incomplete, and the presence of spurious signals limited the dynamic range. To circumvent this problem, we implemented a proximity ligation-based approach, that only leads to a signal when two pre- and postsynaptic markers are sufficiently close. We demonstrate that this approach can be applied to different synaptic marker combinations and can be successfully used for quantification of synapse density in cultures of different maturity stage in healthy or pathological conditions. Thus, the unbiased analysis of synapse labeling and exploitation of resident protein proximity, allows increasing the sensitivity of synapse quantifications in neuronal culture and therefore represents a valuable extension of the analytical toolset for in vitro synapse screens.

scholarly journals 2,7-Bis-(4-Amidinobenzylidene)-Cycloheptan-1-One Dihydrochloride, tPA Stop, Prevents tPA-Enhanced Excitotoxicity Both In Vitro and In Vivo

2004 ◽  
Vol 24 (10) ◽  
pp. 1153-1159 ◽  
Author(s):  
Géraldine Liot ◽  
Karim Benchenane ◽  
Frédéric Léveillé ◽  
José P. López-Atalaya ◽  
Mónica Fernández-Monreal ◽  
...  
Keyword(s):  
Brain Injuries ◽  
Calcium Influx ◽  
Cortical Cell ◽  
Tissue Type ◽  
Type Plasminogen Activator ◽  
Neuroprotective Strategy ◽  
Cerebral Parenchyma ◽  
Cortical Cell Cultures

Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-d-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.

scholarly journals The Interaction of TRAF6 With Neuroplastin Promotes Spinogenesis During Early Neuronal Development

2020 ◽  
Vol 8 ◽  
Author(s):  
Sampath Kumar Vemula ◽  
Ayse Malci ◽  
Lennart Junge ◽  
Anne-Christin Lehmann ◽  
Ramya Rama ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Synapse Formation ◽  
Tumor Necrosis Factor Receptor ◽  
Physical Interaction ◽  
Binding Assays ◽  
C Terminus ◽  
Synapse Density ◽  
Pharmacological Blockade ◽  
Necrosis Factor

Correct brain wiring depends on reliable synapse formation. Nevertheless, signaling codes promoting synaptogenesis are not fully understood. Here, we report a spinogenic mechanism that operates during neuronal development and is based on the interaction of tumor necrosis factor receptor-associated factor 6 (TRAF6) with the synaptic cell adhesion molecule neuroplastin. The interaction between these proteins was predicted in silico and verified by co-immunoprecipitation in extracts from rat brain and co-transfected HEK cells. Binding assays show physical interaction between neuroplastin’s C-terminus and the TRAF-C domain of TRAF6 with a Kd value of 88 μM. As the two proteins co-localize in primordial dendritic protrusions, we used young cultures of rat and mouse as well as neuroplastin-deficient mouse neurons and showed with mutagenesis, knock-down, and pharmacological blockade that TRAF6 is required by neuroplastin to promote early spinogenesis during in vitro days 6-9, but not later. Time-framed TRAF6 blockade during days 6–9 reduced mEPSC amplitude, number of postsynaptic sites, synapse density and neuronal activity as neurons mature. Our data unravel a new molecular liaison that may emerge during a specific window of the neuronal development to determine excitatory synapse density in the rodent brain.

IN VITRO GNAO1-ENCEPHALOPATHY MODEL DEVELOPMENT USING RECOMBINANT ADENO-ASSOCIATED VIRUSES

2020 ◽  
pp. 99-100
Keyword(s):  
Rna Interference ◽  
Model Development ◽  
Neuronal Culture ◽  
Wild Type ◽  
Primary Neuronal Culture ◽  
Neuronal Expression

Our study presents the verification of recombinant adeno-associated viruses on primary neuronal culture for GNAO1-encephalopathy modeling. We demonstrated neuronal expression of transgenic mutant and wild-type forms of the GNAO1. Downregulation by two-fold of the endogenous GNAO1 in neurons was achieved using RNA interference approach.

scholarly journals Contrasting effects of chronic lithium, haloperidol and olanzapine exposure on synaptic clusters in the rat prefrontal cortex

2020 ◽  
Author(s):  
Els F. Halff ◽  
Marie-Caroline Cotel ◽  
Sridhar Natesan ◽  
Richard McQuade ◽  
Chris J. Ottley ◽  
...  
Keyword(s):  
Chronic Exposure ◽  
Antipsychotic Drugs ◽  
Synapse Formation ◽  
Synaptic Dysfunction ◽  
Synaptic Loss ◽  
Marker Proteins ◽  
Synaptic Markers ◽  
Synaptic Pathology ◽  
Cluster Density ◽  
Synapse Density

AbstractThe pathophysiology of the majority of neuropsychiatric disorders, including schizophrenia and mood disorders, involves synaptic dysfunction and/or loss, manifesting as lower levels of several presynaptic and postsynaptic marker proteins. Whether chronic exposure to antipsychotic drugs may contribute to this pattern of synaptic loss remains controversial. In contrast, the mood stabiliser lithium has shown to exhibit neurotrophic actions and is thought to enhance synapse formation. Whilst these data are not unequivocal, they suggest that antipsychotic drugs and lithium have contrasting effects on synapse density. We therefore investigated the effect of chronic exposure to lithium and to two different antipsychotics, haloperidol and olanzapine, on presynaptic Synaptic Vesicle glycoprotein 2A (SV2A) and postsynaptic Neuroligin (NLGN) clusters in the rat frontal cortex. Chronic exposure (28 days) to haloperidol (0.5 mg/kg/d) or olanzapine (7.5 mg/kg/d) had no effect on either SV2A or NLGN clusters and no overall effect on synaptic clusters. In contrast, chronic lithium exposure (2 mmol/L eq./d) significantly increased NLGN cluster density as compared to vehicle, but did not affect either SV2A or total synaptic clusters. These data are consistent with and extend our prior work, confirming no effect of either antipsychotics or lithium on SV2A clustering, but suggest contrasting effects of these drugs on the post-synapse. Although caution needs to be exerted when extrapolating results from animals to patients, these data provide clarity with regard to the effect of antipsychotics and lithium on synaptic markers, thus facilitating discrimination of drug from illness effects in human studies of synaptic pathology in psychiatric disorders.

scholarly journals Effects of Trace Metal Profiles Characteristic for Autism on Synapses in Cultured Neurons

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Simone Hagmeyer ◽  
Katharina Mangus ◽  
Tobias M. Boeckers ◽  
Andreas M. Grabrucker
Keyword(s):  
Hippocampal Neurons ◽  
Metal Ion ◽  
Synapse Formation ◽  
Causative Factor ◽  
Autism Spectrum ◽  
Zn Deficiency ◽  
Protein Levels ◽  
Synapse Density ◽  
Profile Characteristic

Various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders (ASD). Substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as Fe, Cu, Pb, Hg, Se, and Zn may mediate synaptic dysfunction and impair synapse formation and maturation. Here, we performedin vitrostudies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons. We analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers. Moreover, we evaluated whether a biometal profile characteristic for ASD patients influences synapse formation, maturation, and composition regarding NMDA receptor subunits and Shank proteins. Our results show that an ASD like biometal profile leads to a reduction of NMDAR (NR/Grin/GluN) subunit 1 and 2a, as well as Shank gene expression along with a reduction of synapse density. Additionally, synaptic protein levels of GluN2a and Shanks are reduced. Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor. Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.

scholarly journals Glial Glycogen Stores Affect Neuronal Survival during Glucose Deprivation in vitro

1993 ◽  
Vol 13 (1) ◽  
pp. 162-169 ◽  
Author(s):  
Raymond A. Swanson ◽  
Dennis W. Choi
Keyword(s):  
Neuronal Degeneration ◽  
Neuronal Survival ◽  
Cortical Cell ◽  
Glucose Deprivation ◽  
Methionine Sulfoximine ◽  
Protective Effects ◽  
Cortical Cell Cultures ◽  
Energy Dependent ◽  
Glycogen Stores

Glia perform several energy-dependent functions that may aid neuronal survival under pathological conditions. Glycogen is the major energy reserve in brain, and it is localized almost exclusively to astrocytes. Using murine cortical cell cultures containing both glia and neurons, we examined the effect of altered glial glycogen stores on neuronal survival following glucose deprivation. As previously reported, cultures exposed for several hours to media lacking glucose developed widespread neuronal degeneration without glial degeneration. If glial astrocyte glycogen content was increased to 2–3 times control levels by a 24-h pretreatment with 1 μ M insulin or 0.5 m M methionine sulfoximine (MSO), glucose deprivation-induced neuronal degeneration was attenuated. These protective effects were blocked if glycogen levels were reduced back to control levels by a 30-min exposure to 1 m M dibutyryl cyclic AMP or 20 μ M norepinephrine prior to glucose deprivation. Astrocyte glycogen stores may be an important factor influencing neuronal survival under conditions of energy substrate limitation.

scholarly journals Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

2021 ◽  
Vol 22 (11) ◽  
pp. 5968
Author(s):  
Egor A. Turovsky ◽  
Maria V. Turovskaya ◽  
Evgeniya I. Fedotova ◽  
Alexey A. Babaev ◽  
Viktor S. Tarabykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nmda Receptor ◽  
Cortical Neurons ◽  
Target Genes ◽  
Cortical Cell ◽  
Inhibitory System ◽  
Selective Agonist ◽  
Genes Encoding ◽  
Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

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