Abstract
The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.
Highlights
It is well known that the brain consists of hundreds of distinct neuronal cell types [1,2,3] which express different proteins, including receptors, transcription factors, and adhesion molecules that lead to distinct developmental programs, distinct axon outgrowth decisions, distinctPLOS ONE | DOI:10.1371/journal.pone.0140010 October 14, 2015Diversity of Neuronal Cells and Endosomal Nsg1/2 in Primary Cultures circuit formation, distinct excitability, and distinct functions in the circuits
Do different markers commonly used to identify neurons stain the same cells in cultured neurons?
We began our analysis in hippocampal cultures grown for nine days (9 days in vitro = DIV9) by testing the co-expression of three markers for neurons: MAP2, DCX and NeuN
Summary
It is well known that the brain consists of hundreds of distinct neuronal cell types [1,2,3] which express different proteins, including receptors, transcription factors, and adhesion molecules that lead to distinct developmental programs, distinct axon outgrowth decisions, distinctPLOS ONE | DOI:10.1371/journal.pone.0140010 October 14, 2015Diversity of Neuronal Cells and Endosomal Nsg1/2 in Primary Cultures circuit formation, distinct excitability, and distinct functions in the circuits (for example [4,5]). For studies of other cell biological problems (such as axon and dendrite specification and growth, in vitro guidance, polarized trafficking), the particular cell type of an individual cell studied is rarely known, and it is usually assumed that the basic cell biology is identical in all neuronal subtypes. The Benson lab showed that some, but not all, cultured cortical neurons respond to Sema3A by collapsing their growth cones. They discovered that the observed neuron-to-neuron variability was due to celltype differences [9]. The neuron-to-neuron variability frequently encountered in cellular assays might be due to different biology in distinct neuronal cell types
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