Preparation of dissociated mouse primary neuronal cultures from long-term cryopreserved brain tissue

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.

Download Full-text

Neuroinflammatory changes of the normal brain tissue in cured mice following combined radiation and anti-PD-1 blockade therapy for glioma

Scientific Reports ◽

10.1038/s41598-021-84600-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mariano Guardia Clausi ◽

Alexander M. Stessin ◽

Zirun Zhao ◽

Stella E. Tsirka ◽

Samuel Ryu

Keyword(s):

Brain Tissue ◽

Cranial Irradiation ◽

Subcortical White Matter ◽

Glioma Cell Line ◽

Hippocampal Neurogenesis ◽

Normal Brain ◽

Long Term Effects ◽

Normal Brain Tissue ◽

Molecule Inhibitor

AbstractThe efficacy of combining radiation therapy with immune checkpoint inhibitor blockade to treat brain tumors is currently the subject of multiple investigations and holds significant therapeutic promise. However, the long-term effects of this combination therapy on the normal brain tissue are unknown. Here, we examined mice that were intracranially implanted with murine glioma cell line and became long-term survivors after treatment with a combination of 10 Gy cranial irradiation (RT) and anti-PD-1 checkpoint blockade (aPD-1). Post-mortem analysis of the cerebral hemisphere contralateral to tumor implantation showed complete abolishment of hippocampal neurogenesis, but neural stem cells were well preserved in subventricular zone. In addition, we observed a drastic reduction in the number of mature oligodendrocytes in the subcortical white matter. Importantly, this observation was evident specifically in the combined (RT + aPD-1) treatment group but not in the single treatment arm of either RT alone or aPD-1 alone. Elimination of microglia with a small molecule inhibitor of colony stimulated factor-1 receptor (PLX5622) prevented the loss of mature oligodendrocytes. These results identify for the first time a unique pattern of normal tissue changes in the brain secondary to combination treatment with radiotherapy and immunotherapy. The results also suggest a role for microglia as key mediators of the adverse treatment effect.

Download Full-text

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

Download Full-text

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.

Download Full-text