Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons

Salazar, Ivan L.; Mele, Miranda; Caldeira, Margarida V.; Costa, Raimundo Bezerra da; Correia, Bárbara; Frisari, Simone; Duarte, Cristiana

doi:10.21769/bioprotoc.2551

Cited by 14 publications

(11 citation statements)

References 6 publications

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“…Hippocampal neurons were derived from the brain of newborn (1–3 days old) Sprague–Dawley rats according to the protocol [ 29 ]. Briefly, the hippocampus was dissected and separated from meninges and surrounding tissue prior to enzymatic digestion with 0.25% w/v trypsin for 10 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance

2021

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Numerous nanostructured synthetic scaffolds mimicking the architecture of the natural extracellular matrix (ECM) have been described, but the polymeric nanofibers comprising the scaffold were substantially thicker than the natural collagen nanofibers of neural ECM. Here, we report neuron growth on electrospun scaffolds of nylon-4,6 fibers with an average diameter of 60 nm, which closely matches the diameter of collagen nanofibers of neural ECM, and compare their properties with the scaffolds of thicker 300 nm nanofibers. Previously unmodified nylon was not regarded as an independent nanostructured matrix for guided growth of neural cells; however, it is particularly useful for ultrathin nanofiber production. We demonstrate that, while both types of fibers stimulate directed growth of neuronal processes, ultrathin fibers are more efficient in promoting and accelerating neurite elongation. Both types of scaffolds also improved synaptogenesis and the formation of connections between hippocampal neurons; however, the mechanisms of interaction of neurites with the scaffolds were substantially different. While ultrathin fibers formed numerous weak immature β1-integrin-positive focal contacts localized over the entire cell surface, scaffolds of submicron fibers formed β1-integrin focal adhesions only on the cell soma. This indicates that the scaffold nanotopology can influence focal adhesion assembly involving various integrin subunits. The fabricated nanostructured scaffolds demonstrated high stability and resistance to biodegradation, as well as absence of toxic compound release after 1 month of incubation with live cells in vitro. Our results demonstrate the high potential of this novel type of nanofibers for clinical application as substrates facilitating regeneration of nervous tissue.

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Section: Methodsmentioning

confidence: 99%

ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance

2021

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“…Primary cultures of hippocampal neurons were prepared from E18-E19 rat embryos from pregnant dams [20,21]. The hippocampi were dissected from the brain, digested with trypsin and the dissociated cells after sufficient washes were seeded on-to plates, which were precoated with poly-D-lysine (100 µg/ml) and laminin (1µg/ml).…”

Section: Primary Neuronal Cultures and Transfectionmentioning

confidence: 99%

Novel microRNAs targeting NMDA receptor subunits in animal models of schizophrenia

Gunasekaran

Jacob

Omkumar

2021

Preprint

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N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3`UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia- pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. MiR-296-3p, miR-148b-5p and miR-137 levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of neuregulin 1 (NRG1), BDNF and CaMKIIα, genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

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“… Centrifuge the flow-through at 180 × g for 5 min at 4°C with max acceleration and brake. If the supernatant is cloudy, repeat resuspension and centrifugation ( Salazar et al., 2017 ). Discard the supernatant.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…This might occur after severe cell damage when DNA causes cells and other debris to aggregate into large clumps. Images of non-dissociated aggregates are available in an established protocol ( Salazar et al., 2017 ).…”

Section: Troubleshootingmentioning

confidence: 99%

Protocol for Co-culture of Microglia with Axons

Fujita

Yamashita

2020

STAR Protocols

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Summary Microglia, the resident immune cells of the central nervous system, accumulate along axons and support neuronal survival during the early postnatal period. Although methods have been developed to isolate microglia or neurons, the procedures for co-culture of these cells can inadvertently affect the interactions between them. Here, we describe a protocol to investigate the accumulation of microglia toward neuronal axons using axon isolation culture devices. This approach is useful for modeling neuron-glia associations. For complete details on the use and execution of this protocol, please refer to Ueno et al. (2013) and Fujita et al. (2020) .

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Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons

Cited by 14 publications

References 6 publications

ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance

ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance

Novel microRNAs targeting NMDA receptor subunits in animal models of schizophrenia

Protocol for Co-culture of Microglia with Axons

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