Abstract
Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3–5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16–18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.
Highlights
A full understanding of the structure, function, and development of the mammalian central nervous system (CNS) is necessary to develop treatments that could allow successful regeneration of the adult injured or degenerated tissue
Since opossums have lower body temperature than most placental mammals and slightly lower than other marsupials (Harder et al, 1996), their primary cultures have been maintained at 32◦C, as previously described for organotypic cultures derived from developing opossum cortex (Puzzolo and Mallamaci, 2010)
We have shown that long-term primary neuronal cultures can be successfully obtained from neonatal opossums (M. domestica) and that with the use of the animals of different postnatal age, as well of different procedures and media, the enrichment of different CNS cell types, such as neurons or RGCc, can be obtained
Summary
A full understanding of the structure, function, and development of the mammalian central nervous system (CNS) is necessary to develop treatments that could allow successful regeneration of the adult injured or degenerated tissue. Mammalian developing CNS is challenging to study because of the extreme complexity of the dynamic events involved, including cell proliferation, migration, and differentiation. It emerged that neurodegenerative diseases often originate during development, despite late onset (Schaefers and Teuchert-Noodt, 2016). Dissociated primary neuronal cultures represent an excellent in vitro tool to study CNS development, as well as neuronal maturation and functional activity, at the single-cell level and at the network scale These cultures allow us to gain mechanistic insights in a simplified but more controlled context, compared to in vivo conditions (Beaudoin et al, 2012; Humpel, 2015). The studies on primary neuronal cultures contributed to many fundamental discoveries regarding development such as neuronal polarization, neurite outgrowth, axon guidance (pathfinding), M. domestica Primary Neuronal Cultures synaptogenesis and neuronal network formation, activity and maturation, recapitulating in vitro many aspects that occur in vivo (Cáceres et al, 2012; Kaech et al, 2012)
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