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      Culturing primary neurons from rat hippocampus and cortex

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          Abstract

          Primary neurons from rodent brain hippocampus and cortex have served as important tools in biomedical research over the years. However, protocols for the preparation of primary neurons vary, which often lead to conflicting results. This report provides a robust and reliable protocol for the production of primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-neuronal cells. The neurons were grown in serum-free media and maintained for several weeks without any additional feeder cells. The neuronal cultures maintained according to this protocol differentiate and by 3 weeks develop extensive axonal and dendritic branching. The cultures produced by this method show excellent reproducibility and can be used for histological, molecular and biochemical methods.

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          Most cited references 22

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          Culturing hippocampal neurons.

          We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2-3 h.
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            Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

            We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.
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              In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.

              Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.
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                Author and article information

                Contributors
                Journal
                Neuronal Signal
                Neuronal Signal
                ns
                Neuronal Signaling
                Portland Press Ltd.
                2059-6553
                June 2019
                26 April 2019
                : 3
                : 2
                Affiliations
                Neuroscience Center, Helsinki Institute of Life Science HiLIFE, University of Helsinki, Helsinki 00290, Finland
                Author notes
                Correspondence: Eero Castrén ( eero.castren@ 123456helsinki.fi )
                Article
                NS20180207
                10.1042/NS20180207
                7363313
                © 2019 The Author(s).

                This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).

                Page count
                Pages: 10
                Product
                Categories
                Signaling
                Biochemical Techniques & Resources
                Neuroscience
                Research Articles

                cerebral cortex, hippocampus, primary neuron

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