title: Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).
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.
authors.affiliation: University of Virginia, Department of Neuroscience, 409 Lane Road, Charlottesville, VA, 22908, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Digilio
authors.affiliation: University of Virginia, Department of Neuroscience, 409 Lane Road, Charlottesville, VA, 22908, United States of America.
authors.forename: Chan Choo
authors.initials: CC
authors.lastname: Yap
authors.affiliation: University of Virginia, Department of Neuroscience, 409 Lane Road, Charlottesville, VA, 22908, United States of America.
authors.forename: Bettina
authors.initials: B
authors.lastname: Winckler
mesh_terms: D000818:Animals; D015415:Biomarkers; D002478:Cells, Cultured; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009928:Organ Specificity; D017966:Pyramidal Cells / Q000166:cytology / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D000098551:Prospero-Related Homeobox 1 Protein
chemical_list: C000596384:BCL11B protein, rat; D015415:Biomarkers; D018398:Homeodomain Proteins; D036961:Matrix Attachment Region Binding Proteins; D009419:Nerve Tissue Proteins; C461339:Nsg1 protein, rat; D012097:Repressor Proteins; C586446:SATB2 protein, rat; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000098551:Prospero-Related Homeobox 1 Protein; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2