Atomic View of an Amyloid Dodecamer Exhibiting Selective Cellular Toxic Vulnerability in Acute Brain Slices

Transport of histidine, valine, or lysine into rat brain slices and across the blood-brain barrier (BBB) was determined in the presence of atypical nonprotein amino acids. Competitors of histidine and valine transport in slices were large neutral amino acids including norleucine, norvaline, alpha-aminooctanoate, beta-methylphenylalanine, and alpha-aminophenylacetate. Less effective were aromatic amino acids with ring substituents; ineffective were basic amino acids and omega-amino isomers of norleucine and aminooctanoate. Lysine transport was moderately depressed by homoarginine or ornithine plus arginine; large neutral amino acids were also similarly inhibitory. Histidine or valine transport across the BBB was also strongly inhibited by large neutral amino acids that were the most effective competitors in the slices (norvaline, norleucine, alpha-aminooctanoate, and alpha-aminophenylacetate); homoarginine and 8-aminooctanoate were ineffective. Homoarginine, ornithine, and arginine almost completely blocked lysine transport, but the large neutral amino acids were barely inhibitory. When rats were fed a single meal containing individual atypical large neutral amino acids or homoarginine, brain pools of certain large neutral amino acids or of arginine and lysine, respectively, were depleted.

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Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices

Brain Research ◽

10.1016/s0006-8993(97)00632-x ◽

1997 ◽

Vol 767 (1) ◽

pp. 72-80 ◽

Cited By ~ 24

Author(s):

Marcel T.G de Jeu ◽

Cyriel M.A Pennartz

Keyword(s):

Patch Clamp ◽

Functional Characterization ◽

Brain Slices ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp ◽

Clamp Study

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Oxytocin Modulation of Maternal Behavior and Its Association With Immunological Activity in Rats With Cesarean Delivery

ASN NEURO ◽

10.1177/17590914211014731 ◽

2021 ◽

Vol 13 ◽

pp. 175909142110147

Author(s):

Tong Li ◽

Shu-Wei Jia ◽

Dan Hou ◽

Xiaoran Wang ◽

Dongyang Li ◽

...

Keyword(s):

Cesarean Delivery ◽

Maternal Behavior ◽

Inflammatory Cytokine ◽

Adrenocorticotropic Hormone ◽

Brain Slices ◽

Relative Weight ◽

Resting Membrane Potential ◽

Cytokine Release ◽

Immunological Activity ◽

Fos Expression

Oxytocin (OT), a neuropeptide produced in the supraoptic (SON) and paraventricular (PVN) nuclei, is not only essential for lactation and maternal behavior but also for normal immunological activity. However, mechanisms underlying OT regulation of maternal behavior and its association with immunity around parturition, particularly under mental and physical stress, remain unclear. Here, we observed effects of OT on maternal behavior in association with immunological activity in rats after cesarean delivery (CD), a model of reproductive stress. CD significantly reduced maternal interests to the pups throughout postpartum day 1-8. On postpartum day 5, CD decreased plasma OT levels and thymic index but increased vasopressin, interleukin (IL)-1β, IL-6 and IL-10 levels. CD had no significant effect on plasma adrenocorticotropic hormone and corticosterone levels. In the hypothalamus, CD decreased corticotropin-releasing hormone contents in the PVN but increased OT contents in the PVN and SON and OT release from hypothalamic implants. CD also increased c-Fos expression, particularly in the cytoplasm of OT neurons. Lastly, CD depolarized resting membrane potential and increased spike width while increasing the variability of the firing rate of OT neurons in brain slices. Thus, CD can increase hypothalamic OT contents and release but reduce pituitary release of OT into the blood, which is associated with depressive-like maternal behavior, increased inflammatory cytokine release and decreased relative weight of the thymus.

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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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Comparative analysis of spreading depolarizations in brain slices exposed to osmotic or metabolic stress

BMC Neuroscience ◽

10.1186/s12868-021-00637-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Rita Frank ◽

Ferenc Bari ◽

Ákos Menyhárt ◽

Eszter Farkas

Keyword(s):

Osmotic Stress ◽

Tissue Injury ◽

Brain Slices ◽

Field Potential ◽

Metabolic Stress ◽

Glucose Deprivation ◽

Optical Signal ◽

Light Illumination ◽

Glass Capillary ◽

Experimental Conditions

Abstract Background Recurrent spreading depolarizations (SDs) occur in stroke and traumatic brain injury and are considered as a hallmark of injury progression. The complexity of conditions associated with SD in the living brain encouraged researchers to study SD in live brain slice preparations, yet methodological differences among laboratories complicate integrative data interpretation. Here we provide a comparative evaluation of SD evolution in live brain slices, in response to selected SD triggers and in various media, under otherwise standardized experimental conditions. Methods Rat live coronal brain slices (350 μm) were prepared (n = 51). Hypo-osmotic medium (Na+ content reduced from 130 to 60 mM, HM) or oxygen-glucose deprivation (OGD) were applied to cause osmotic or ischemic challenge. Brain slices superfused with artificial cerebrospinal fluid (aCSF) served as control. SDs were evoked in the control condition with pressure injection of KCl or electric stimulation. Local field potential (LFP) was recorded via an intracortical glass capillary electrode, or intrinsic optical signal imaging was conducted at white light illumination to characterize SDs. TTC and hematoxylin-eosin staining were used to assess tissue damage. Results Severe osmotic stress or OGD provoked a spontaneous SD. In contrast with SDs triggered in aCSF, these spontaneous depolarizations were characterized by incomplete repolarization and prolonged duration. Further, cortical SDs under HM or OGD propagated over the entire cortex and occassionally invaded the striatum, while SDs in aCSF covered a significantly smaller cortical area before coming to a halt, and never spread to the striatum. SDs in HM displayed the greatest amplitude and the most rapid propagation velocity. Finally, spontaneous SD in HM and especially under OGD was followed by tissue injury. Conclusions While the failure of Na+/K+ ATP-ase is thought to impair tissue recovery from OGD-related SD, the tissue swelling-related hyper excitability and the exhaustion of astrocyte buffering capacity are suggested to promote SD evolution under osmotic stress. In contrast with OGD, SD propagating under hypo-osmotic condition is not terminal, yet it is associated with irreversible tissue injury. Further investigation is required to understand the mechanistic similarities or differences between the evolution of SDs spontaneously occurring in HM and under OGD.

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The Entorhinal Cortical Alvear Pathway Differentially Excites Pyramidal Cells and Interneuron Subtypes in Hippocampal CA1

Cerebral Cortex ◽

10.1093/cercor/bhaa359 ◽

2020 ◽

Author(s):

Karen A Bell ◽

Rayne Delong ◽

Priyodarshan Goswamee ◽

A Rory McQuiston

Keyword(s):

Brain Slices ◽

Pyramidal Cells ◽

Excitatory Input ◽

Theta Burst Stimulation ◽

Ca1 Pyramidal Cells ◽

Whole Cell Patch Clamp ◽

Hippocampal Ca1 ◽

Physiological Impact ◽

Theta Burst ◽

Synaptic Responses

Abstract The entorhinal cortex alvear pathway is a major excitatory input to hippocampal CA1, yet nothing is known about its physiological impact. We investigated the alvear pathway projection and innervation of neurons in CA1 using optogenetics and whole cell patch clamp methods in transgenic mouse brain slices. Using this approach, we show that the medial entorhinal cortical alvear inputs onto CA1 pyramidal cells (PCs) and interneurons with cell bodies located in stratum oriens were monosynaptic, had low release probability, and were mediated by glutamate receptors. Optogenetic theta burst stimulation was unable to elicit suprathreshold activation of CA1 PCs but was capable of activating CA1 interneurons. However, different subtypes of interneurons were not equally affected. Higher burst action potential frequencies were observed in parvalbumin-expressing interneurons relative to vasoactive-intestinal peptide-expressing or a subset of oriens lacunosum-moleculare (O-LM) interneurons. Furthermore, alvear excitatory synaptic responses were observed in greater than 70% of PV and VIP interneurons and less than 20% of O-LM cells. Finally, greater than 50% of theta burst-driven inhibitory postsynaptic current amplitudes in CA1 PCs were inhibited by optogenetic suppression of PV interneurons. Therefore, our data suggest that the alvear pathway primarily affects hippocampal CA1 function through feedforward inhibition of select interneuron subtypes.

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TAMI-28. DIFFERENTIAL MIGRATION MECHANICS AND IMMUNE RESPONSES OF GLIOMA SUBTYPES

Neuro-Oncology ◽

10.1093/neuonc/noaa215.916 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii219-ii219

Author(s):

Ghaidan Shamsan ◽

Chao Liu ◽

Brooke Braman ◽

Susan Rathe ◽

Aaron Sarver ◽

...

Keyword(s):

Tumor Cells ◽

Immune Cell ◽

Molecular Subtypes ◽

Ex Vivo ◽

Brain Slices ◽

Traction Force ◽

Genetic Alterations ◽

Sleeping Beauty ◽

Brain Parenchyma ◽

Biophysical Modeling

Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.

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Quantitative measurement of reactive oxygen species in ex vivo mouse brain slices

STAR Protocols ◽

10.1016/j.xpro.2021.100332 ◽

2021 ◽

Vol 2 (1) ◽

pp. 100332

Author(s):

Chirag Vasavda ◽

Solomon H. Snyder ◽

Bindu D. Paul

Keyword(s):

Reactive Oxygen Species ◽

Mouse Brain ◽

Quantitative Measurement ◽

Ex Vivo ◽

Brain Slices ◽

Reactive Oxygen ◽

Oxygen Species

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A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽

10.1093/function/zqab012 ◽

2021 ◽

Vol 2 (3) ◽

Cited By ~ 1

Author(s):

Nelly Redolfi ◽

Elisa Greotti ◽

Giulia Zanetti ◽

Tino Hochepied ◽

Cristina Fasolato ◽

...

Keyword(s):

Transgenic Mouse ◽

Fluorescent Protein ◽

Ex Vivo ◽

Brain Slices ◽

Resonance Energy ◽

Mouse Line ◽

Isolated Cells ◽

Genomic Locus ◽

Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

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