scholarly journals High-dose Propofol Triggers Short-term Neuroprotection and Long-term Neurodegeneration in Primary Neuronal Cultures from Rat Embryos

2009 ◽  
Vol 37 (3) ◽  
pp. 680-688 ◽  
Author(s):  
M Berns ◽  
L Seeberg ◽  
M Schmidt ◽  
T Kerner
Keyword(s):  
Cell Viability ◽  
Cortical Neurons ◽  
Neuroprotective Effect ◽  
Wistar Rat ◽  
Neuronal Cultures ◽  
High Dose ◽  
Receptor Antagonists ◽  
Primary Neuronal Cultures ◽  
Rat Embryos ◽  
High Doses

This study investigated the effects of propofol on primary neuronal cultures from rat embryos. Primary cortical neuronal cultures were prepared from Wistar rat embryos (E18). The viability of cells exposed to 0.01, 0.1 or 1 mg/ml propofol for up to 48 h was assessed using a methyltetrazolium assay. In order to evaluate the role of γ-aminobutyric acid-A (GABAA) receptors, cells were also pre-incubated with the GABAA-receptor antagonists, gabazine and picrotoxin. Propofol at a concentration of 1 mg/ml significantly reduced cell viability after 12 h. In contrast, this concentration led to a significant increase in cell viability at 3 and 6 h. The GABAA-receptor antagonists did not influence the neurodegenerative effect of propofol but abolished its neuroprotective effect. DNA fragmentation as a marker of apoptosis was elevated after 24 h propofol treatment. These results confirm that high doses of propofol can cause GABAA-receptor triggered neuroprotection and a subsequent time-dependent, but GABAA-independent, neurodegeneration in primary cortical neurons.

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

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.

Assessment of Cell Viability in Primary Neuronal Cultures

2008 ◽  
Vol 44 (1) ◽  
Author(s):  
Mandar A. Aras ◽  
Karen A. Hartnett ◽  
Elias Aizenman
Keyword(s):  
Cell Viability ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures

Effect of vitamin D treatment on VDR expression in primary cerebral cortical cells in induced oxidative stress

2020 ◽  
pp. 1-10
Author(s):  
Ebtesam Alsulami ◽  
Majed Alokail ◽  
Amani Alghamedi ◽  
Abir Alamro ◽  
Samina Haq
Keyword(s):  
Oxidative Stress ◽  
Vitamin D ◽  
Vitamin D3 ◽  
Cultured Cells ◽  
Neuroprotective Effect ◽  
Sandwich Elisa ◽  
Stress Protein ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Peripheral Tissues

BACKGROUND: In addition to calcium and phosphate homeostasis in peripheral tissues; vitamin D performs a neuroprotection role in the nervous system. The neuroprotective actions of vitamin D include: increasing vitamin D receptor (VDR) expression, control glutathione synthesis and nitric oxide synthase activity and induce neurotrophins such as nerve growth factor (NGF). VDR mediates cellular actions, and biological responses of the vitamin D. OBJECTIVE: To study the effect of VDR and NGF expression levels by vitamin D3 treatment in induced oxidative stress in primary cortical neuronal cultures. METHOD: Primary neuronal cultures were set up from the cortex region of neonatal rat’s brain. They were cultured for up to 72 h in the presence of 0.25μg/ml vitamin D3. These cells were exposed to 0.5 mM H2O2 for two hours before collecting cell pellet and medium for biochemical assays. Control and H2O2 treated cells were cultured in the absence of vitamin D3 treatment. Sandwich ELISA was used to study NGF expression. Western blotting and Immunofluorescence of cultured cells were used to estimate the expression of VDR. RESULTS: Vitamin D3 treatment increased more significantly (P <  0.001) NGF levels with and without induced oxidative stress. Protein expression studies confirmed the positive correlation between VDR expression and vitamin D3 treatment after 72 h in culture. Moreover, pre-treating the cells with vitamin D3 before H2O2 exposure significantly increase (P <  0.05) VDR expression in comparison with the cells exposed to H2O2 alone. CONCLUSION: The neuroprotective effect of vitamin D3 against oxidative stress could be through up-regulating VDR and NGF levels.

scholarly journals Development of a High-Throughput AlphaScreen Assay for Modulators of Synapsin I Phosphorylation in Primary Neurons

2013 ◽  
Vol 19 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Betty Chan ◽  
Jeffrey R. Cottrell ◽  
Bing Li ◽  
Kelley C. Larson ◽  
Crystle J. Ashford ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Critical Role ◽  
Neuronal Cultures ◽  
Synapsin I ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Small Molecule Modulators

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.

scholarly journals Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells

2017 ◽  
Vol 11 (1) ◽  
pp. 39-47
Author(s):  
Monika Berns ◽  
Anna Christine Wolter ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thoralf Kerner
Keyword(s):  
Cell Viability ◽  
Neuronal Cells ◽  
Neuronal Cultures ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Amino Butyric Acid ◽  
Wide Range ◽  
Primary Neuronal Cells ◽  
The Impact

Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 - 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γ-amino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure.

Neuroprotective effect of weak static magnetic fields in primary neuronal cultures

Neuroscience ◽  
2014 ◽  
Vol 278 ◽  
pp. 313-326 ◽  
Author(s):  
M. Ben Yakir-Blumkin ◽  
Y. Loboda ◽  
L. Schächter ◽  
J.P.M. Finberg
Keyword(s):  
Magnetic Fields ◽  
Neuroprotective Effect ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Static Magnetic Fields

scholarly journals The protective effects of melatonin in high glucose environment by alleviating autophagy and apoptosis on primary cortical neurons

2021 ◽  
Author(s):  
Xiong Lijiao ◽  
Song Liu ◽  
Chaoming Liu ◽  
Tianting Guo ◽  
Zhihua Huang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Protective Effect ◽  
Cell Viability ◽  
High Glucose ◽  
Cortical Neurons ◽  
Neuroprotective Effect ◽  
Control Group ◽  
Primary Cortical Neurons ◽  
Apoptosis Rate ◽  
Glucose Group

Abstract Cognitive dysfunction has been regarded as a complication of diabetes. Melatonin shows a neuroprotective effect on various neurological diseases. However, it’s protective effect on cortical neurons in high glucose environment has not been reported. Our present study aims to observe the protective effect of melatonin on rat cortical neurons and its relationship with autophagy in high glucose environment. The rat primary cortical neurons damaged model was induced by high glucose. The CCK-8, flow cytometry, Western Blot and immunofluorescence methods were used to examine the cell viability, apoptosis rate and proteins expression. Our results showed that there were no differences in cell viability, apoptosis rate, and protein expression among the control MLT and mannitol group. The cell viability of the glucose group was significantly lower than that of the control group, and the apoptosis rate of the glucose group was significantly higher than that of the control group. Compared with the glucose group, the glucose + melatonin group showed a significant increase in cell viability and a notable decrease in apoptosis rate. Melatonin concentration of 0.1-1 mmol/L can significantly reduce the injury of cortical neurons by high glucose. Compared with the control group, the glucose group showed a significant reduction of Bcl-2 protein expression, while remarkable elevations of Bax, caspase-3, Beclin-1 and LC3B levels. The neurons pre-administered with melatonin obtained significantly reversed these changes induced by high glucose. The phosphorylation levels of Akt and mTOR in the glucose group were significantly lower than those in the control group, were significantly increased in the glucose + MLT group compared with the glucose group. These data indicated that melatonin has a neuroprotective effect on cortical neurons under high glucose environment, which may work by activating Akt/mTOR pathway and following the down-regulation of autophagy.

Assessment of Cell Viability in Primary Neuronal Cultures

2000 ◽  
Vol 13 (1) ◽  
Author(s):  
Howard S. Ying ◽  
Frank J. Gottron ◽  
Dennis W. Choi
Keyword(s):  
Cell Viability ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures

scholarly journals Fibrillar α-synuclein toxicity depends on functional lysosomes

2020 ◽  
Vol 295 (51) ◽  
pp. 17497-17513
Author(s):  
Stephanie J. Guiney ◽  
Paul A. Adlard ◽  
Peng Lei ◽  
Celeste H. Mawal ◽  
Ashley I. Bush ◽  
...  
Keyword(s):  
Cell Line ◽  
Cortical Neurons ◽  
Enzyme Inhibitors ◽  
High Dose ◽  
Bafilomycin A1 ◽  
Cell Death Pathway ◽  
Lysosomal Cysteine Protease ◽  
Dependent Cell ◽  
High Doses

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

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