Long‐Lived Organotypic Slice Culture Model of the Rat Basolateral Amygdala

2021 ◽  
Vol 1 (10) ◽  
Author(s):  
Sheldon D. Michaelson ◽  
Taylor M. Müller ◽  
Maria Bompolaki ◽  
Ana Pamela Miranda Tapia ◽  
Heika Silveira Villarroel ◽  
...  
Keyword(s):  
Basolateral Amygdala ◽  
Slice Culture ◽  
Organotypic Slice Culture ◽  
Culture Model

scholarly journals Contribution of NPY Y5 Receptors to the Reversible Structural Remodeling Of Basolateral Amygdala Dendrites in Male Rats Associated with NPY-mediated Stress Resilience

2019 ◽  
Author(s):  
Sheldon D. Michaelson ◽  
Ana Pamela Miranda Tapia ◽  
Amanda McKinty ◽  
Heika Silveira Villarroel ◽  
James P. Mackay ◽  
...  
Keyword(s):  
Neuropeptide Y ◽  
Structural Changes ◽  
Basolateral Amygdala ◽  
Excitatory Input ◽  
Male Rats ◽  
Repeated Treatment ◽  
Slice Culture ◽  
Stress Resilience ◽  
Organotypic Slice Culture ◽  
Vulnerability To Stress

ABSTRACTEndogenous neuropeptide Y (NPY) and corticotrophin-releasing factor (CRF) modulate the responses of the Basolateral amygdala (BLA) to stress, and are associated respectively with the development of stress resilience and vulnerability. We characterized the persistent effects of repeated NPY and CRF treatment on the structure and function of BLA principal neurons (PN) in a novel organotypic slice culture (OTC) model of rat BLA, and examined the contributions of specific NPY receptor subtypes to these neural and behavioral effects. In BLA principal neurons within the OTCs, repeated NPY treatment caused persistent attenuation of excitatory input and induced dendritic hypotrophy via Y5 receptors; conversely, CRF increased excitatory input and induced hypertrophy of BLA PNs. Repeated treatment of OTCs with NPY followed by an identical treatment with CRF, or vice versa inhibited or reversed all structural changes in OTCs. These structural responses to NPY or CRF required calcineurin or CaMKII, respectively. Finally, repeated intra-BLA injections of NPY or a Y5 receptor agonist increased social interaction and recapitulated structural changes in BLA neurons seen in OTCs, while a Y5 receptor antagonist prevented NPY’s effects both on behavior and on structure. These results implicate the Y5 receptor in the long-term, anxiolytic-like effects of NPY in the BLA, consistent with an intrinsic role in stress buffering, and highlight a remarkable mechanism by which BLA neurons may adapt to different levels of stress. Moreover, BLA OTCs offer a robust model to study mechanisms associated with resilience and vulnerability to stress in BLA.Significance StatementWithin the basolateral amygdala (BLA), Neuropeptide Y (NPY) is associated with buffering the neural stress response induced by CRF, and promoting stress resilience. We used a novel organotypic slice culture (OTC) model of BLA, complemented with in vivo studies, to examine the cellular mechanisms associated with the actions of NPY. In OTCs, repeated NPY treatment reduces the complexity of the dendritic extent of anxiogenic BLA principal neurons, making them less excitable. NPY, via activation of Y5 receptors, additionally inhibits and reverses the increases in dendritic extent and excitability induced by the stress hormone, corticotropin releasing factor (CRF). This NPY-mediated neuroplasticity indicates that resilience or vulnerability to stress may thus involve neuropeptide-mediated dendritic remodeling in BLA PNs.

scholarly journals An organotypic slice culture model of chronic white matter injury with maturation arrest of oligodendrocyte progenitors

2011 ◽  
Vol 6 (1) ◽  
pp. 46 ◽  
Author(s):  
Justin M Dean ◽  
Art Riddle ◽  
Jennifer Maire ◽  
Kelly D Hansen ◽  
Marnie Preston ◽  
...  
Keyword(s):  
White Matter ◽  
White Matter Injury ◽  
Slice Culture ◽  
Maturation Arrest ◽  
Oligodendrocyte Progenitors ◽  
Organotypic Slice Culture ◽  
Culture Model

scholarly journals TMOD-26. ESTABLISHING A PATIENT-DERIVED, IN-VITRO ORGANOTYPIC SLICE CULTURE MODEL OF GBM

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
Liam Rappoldt ◽  
Adrienne Weeks ◽  
Rodney Ouellete ◽  
Jeremy Roy ◽  
Catherine Taylor ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Laser Scanning ◽  
Tumour Microenvironment ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Slice Culture ◽  
Organotypic Cultures ◽  
Organotypic Slice Culture ◽  
Culture Model

Abstract Glioblastoma Multiforme (GBM) is the most common primary malignant brain tumour. This tumour is universally fatal with a median survival of 15 months. Driving this pathology is an extremely heterogeneic tumour and complex tumour microenvironment. GBM research is primarily conducted using immortalized or primary cell lines due to their practicality and reproducibility. However, these cell lines do not effectively recapitulate the tumour microenvironment. Mouse models address these shortcomings but are laborious and expensive. We propose to utilize a patient derived organotypic culture model of GBM as an intermediary. We have utilized this model to test genetic manipulation via lentiviral transduction and the feasibility of utilizing this model to understand patient derived extracellular vesicles (EVs). We have sectioned and cultured patient derived organotypic models for 14 days without loss of viability. To determine if these organotypic cultures are amenable to lentiviral manipulation, tissue sections were transduced with far-red fluorescent lentivirus and efficiency determined by confocal laser scanning microscopy (CLSM) and flow cytometry (FC). To determine feasibility as a model for EVs, media obtained from patient-derived organotypic cultures was analyzed by western blot, nanoparticle tracking analysis (NTA), and nanoFlow Cytometry (nFC). In the future these EVs will be compared to those found in patient serum. The model of GBM has been lentivirally transduced to express a far-red fluorescent vector in approximately 15% of the slice, quantified by CLSM and FC. EV-sized particles positive for canonical EV markers have been identified in the media by NTA, nFC and western blot. Using lentiviral-mediated genetic engineering and emerging EV science, this organotypic slice culture models yields exciting utility in GBM research. The established organotypic slice culture model will likely be a valuable tool in the study of GBM biology and EV dynamics.

Recruiting new neurons from the subventricular zone to the rat postnatal cortex: an organotypic slice culture model

2008 ◽  
Vol 27 (5) ◽  
pp. 1051-1060 ◽  
Author(s):  
A. G. Dayer ◽  
B. Jenny ◽  
G. Potter ◽  
M. O. Sauvain ◽  
G. Szabó ◽  
...  
Keyword(s):  
Subventricular Zone ◽  
Slice Culture ◽  
Organotypic Slice Culture ◽  
Culture Model

scholarly journals Organotypic slice culture model demonstrates interneuronal spreading of alpha-synuclein aggregates

2019 ◽  
Author(s):  
Sara Elfarrash ◽  
Nanna Møller Jensen ◽  
Nelson Ferreira ◽  
Cristine Betzer ◽  
Jervis Vermal Thevathasan ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Hippocampal Slice ◽  
Alpha Synuclein ◽  
Genetically Engineered ◽  
Slice Culture ◽  
Lewy Pathology ◽  
Organotypic Slice Culture ◽  
Genetically Engineered Mice ◽  
Culture Model

AbstractHere we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by injection of preformed α-synuclein filaments (PFFs). PFF injection at dentate gyrus templates the endogenous α-synuclein to form aggregates in axons and cell bodies that spread to CA3 and CA1 regions. Aggregates were insoluble and phosphorylated at serine 129, recapitulating Lewy pathology features found in Parkinson’s disease and other synucleinopathies. The spreading of the aggregates were favoring the anterograde direction in the slice model. The model allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using adeno-associated viral (AAV) vector in α-synuclein knockout slices. Processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine 129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated by using viral vectors allow testing of hypotheses on mechanisms involved in formation of α-synuclein aggregates and their prion-like spreading.

scholarly journals Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Sara Elfarrash ◽  
Nanna Møller Jensen ◽  
Nelson Ferreira ◽  
Cristine Betzer ◽  
Jervis Vermal Thevathasan ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Hippocampal Slice ◽  
Alpha Synuclein ◽  
Genetically Engineered ◽  
Slice Culture ◽  
Lewy Pathology ◽  
Organotypic Slice Culture ◽  
Genetically Engineered Mice ◽  
Culture Model

AbstractHere we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson’s disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.

Melatonin acts as antioxidant and pro-oxidant in an organotypic slice culture model of Alzheimer's disease

Neuroreport ◽  
2001 ◽  
Vol 12 (6) ◽  
pp. 1277-1280 ◽  
Author(s):  
Kimberly L. Clapp-Lilly ◽  
Mark A. Smith ◽  
George Perry ◽  
Peggy L. Harris ◽  
Xiongwei Zhu ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Slice Culture ◽  
Organotypic Slice Culture ◽  
Model Of Alzheimer’S Disease ◽  
Culture Model

Isoflurane Prevents Delayed Cell Death in an Organotypic Slice Culture Model of Cerebral Ischemia

2002 ◽  
Vol 96 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Breandan L. Sullivan ◽  
David Leu ◽  
Donald M. Taylor ◽  
Christian S. Fahlman ◽  
Philip E. Bickler
Keyword(s):  
Cell Death ◽  
Cerebral Ischemia ◽  
Dentate Gyrus ◽  
Neuronal Death ◽  
Slice Culture ◽  
Mk 801 ◽  
Organotypic Slice Culture ◽  
Delayed Cell Death ◽  
Culture Model

Background General anesthetics reduce neuronal death caused by focal cerebral ischemia in rodents and by in vitro ischemia in cultured neurons and brain slices. However, in intact animals, the protective effect may enhance neuronal survival for only several days after an ischemic injury, possibly because anesthetics prevent acute but not delayed cell death. To further understand the mechanisms and limitations of volatile anesthetic neuroprotection, the authors developed a rat hippocampal slice culture model of cerebral ischemia that permits assessment of death and survival of neurons for at least 2 weeks after simulated ischemia. Methods Survival of CA1, CA3, and dentate gyrus neurons in cultured hippocampal slices (organotypic slice culture) was examined 2-14 days after 45 min of combined oxygen-glucose deprivation at 37 degrees C (OGD). Delayed cell death was serially measured in each slice by quantifying the binding of propidium iodide to DNA with fluorescence microscopy. Results Neuronal death was greatest in the CA1 region, with maximal death occurring 3-5 days after OGD. In CA1, cell death was 80 +/- 18% (mean +/- SD) 3 days after OGD and was 80-100% after 1 week. Death of 70 +/- 16% of CA3 neurons and 48 +/- 28% of dentate gyrus neurons occurred by the third day after OGD. Both isoflurane (1%) and the N-methyl-D-aspartate antagonist MK-801 (10 microm) reduced cell death to levels similar to controls (no OGD) for 14 days after the injury. Isoflurane also reduced cell death in CA1 and CA3 caused by application of 100 but not 500 microm glutamate. Cellular viability (calcein fluorescence) and morphology were preserved in isoflurane-protected neurons. Conclusions In an in vitro model of simulated ischemia, 1% isoflurane is of similar potency to 10 microm MK-801 in preventing delayed cell death. Modulation of glutamate excitotoxicity may contribute to the protective mechanism.

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