Abstract
Spinal cord interneurons (SpINs) are highly diverse population of neurons that play a significant role in circuit reorganization and spontaneous recovery after spinal cord injury. Regeneration of SpIN axons across rodent spinal injuries has been demonstrated after modification of the environment and neurotrophin treatment, but development of methods to enhance the intrinsic regenerative ability of SpINs is needed. There is a lack of described in vitro models of spinal cord neurons in which to develop new regeneration treatments. For this reason, we developed a new model of mouse primary spinal cord neuronal culture in which to analyze maturation, morphology, physiology, connectivity and regeneration of identified interneurons. Isolated from E14 mice, the neurons mature over 15 days in vitro, demonstrated by expression of maturity markers, electrophysiological patch-clamp recordings, and formation of synapses. The neurons express markers of SpINs, including Tlx3, Lmx1b, Lbx1, Chx10, and Pax2. The neurons demonstrate distinct morphologies and some form perineuronal nets in long-term cultivation. Live neurons in various maturation stages were axotomized, using a 900 nm multiphoton laser and their fate was observed overnight. The percentage of axons that regenerated declined with neuronal maturity. This model of SpINs will be a valuable tool in future regenerative, developmental, and functional studies alongside existing models using cortical or hippocampal neurons.
Highlights
The intrinsic regeneration capacity of mature mammalian central nervous system (CNS) is poor
The composition of the culture during cultivation was analyzed using immunocytochemistry; maturity of the neurons was analyzed by immunocytochemistry and patch-clamp, and regenerative capacity of neurons was established by laser axotomy
We found that during cultivation, vesicular glutamate transporter 1 (VGLUT1), Homer1, and vesicular GABA transporter (VGAT) colocalized in similar ratios, meaning that in immature and mature cultures, the same fraction of these synaptic markers did not colocalize with their counterpart synaptic marker
Summary
The intrinsic regeneration capacity of mature mammalian central nervous system (CNS) is poor. The extracellular environment of the CNS is not favorable for axon outgrowth due to production of growth-inhibiting molecules such as NogoA and CSPGs from glial scars surrounding the damaged tissue (Shen et al, 2009; Schwab and Strittmatter, 2014; Uyeda and Muramatsu, 2020), and there is a lack of some necessary growth factors that provide trophic support for neurons and act as chemoattractants for axons (Blesch et al, 2012; Anderson et al, 2018). Another important limiting factor is the intrinsic loss of regenerative ability. A high level of intrinsic regeneration ability is present in immature neurons but ceases abruptly with maturation (Nicholls and Saunders, 1996; Lu et al, 2014; Koseki et al, 2017)
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