Corticospinal neurons respond differentially to neurotrophins and myelin‐associated glycoprotein in vitro

Abstract

Elucidating the mechanisms that regulate the survival and outgrowth of corticospinal tract (CST) neurons and other CNS tracts will be a key component in developing novel approaches for the treatment of central nervous system (CNS) disorders, including stroke, spinal cord injury (SCI), and motor neuron disease (MND). However, the in vivo complexities of these diseases make a systematic evaluation of potential therapeutics that directly affect corticospinal regeneration or survival very challenging. Here, we use Thy1.2 transgenic mice expressing yellow fluorescent protein (YFP) in postnatal day 8 (P8) corticospinal neurons, as a source of CST neurons that have already established synapses in the spinal cord, to assess factors that influence neurite outgrowth and survival of axotomized CST neurons. After culture, YFP-positive corticospinal neurons represent an enriched neuronal population over other glia and interneurons, survive, and extend processes over time. YFP-positive CST neurons also continue to express the corticospinal markers CTIP2 and Otx1. CST neurons display different degrees of axon extension, dendritic branch length and elaboration, and neurite elongation in response to neurotrophin-3 and ciliary neurotrophic factor, and an inhibitory outgrowth response when cultured on myelin-associated glycoprotein. Some CST neurons are lost with extended culture, which provides a baseline from which we can also assess factors that enhance CST neuron survival. This assay thus allows us to assess independent aspects of CST axonal and dendritic outgrowth kinetics, which allows for the rapid and sensitive investigation of new therapies to address corticospinal neuron outgrowth in the context of CNS injury and neurodegenerative disorders.

BDNF/trkB Induction of Calcium Transients through Cav2.2 Calcium Channels in Motoneurons Corresponds to F-actin Assembly and Growth Cone Formation on β2-Chain Laminin (221)

“…However, soluble receptor cytokine-like factor 1 (CLF) and cardiotrophin-like cytokine (CLC) represent another ligand for CNTFR, in particular during development, also conveying motoneuron survival via gp130, LIFR and STAT-3 signaling pathways (Elson et al, 2000 ; Forger et al, 2003 ). In corticospinal motoneurons, BDNF mediates axonal arborization and branching, whereas IGF-1 and CNTF induce axon growth (Ozdinler and Macklis, 2006 ; Richter and Roskams, 2009 ). In line with these observations, application of CNTF or IGF-1 ameliorated the ALS-afflicted pmn phenotype (Sendtner et al, 1992 ; Bommel et al, 2002 ; Jablonka et al, 2011 ; Selvaraj et al, 2012 ).…”

Effects of the neurotrophic factors BDNF, NT-3, and FGF2 on dissociated neurons of the cochlear nucleus

“…The analyses were carried out after 8 days in culture because time can be an important factor for the examination of primary cultured neuronal cells. *P<0.05; **P<0.01. elongation and branching are additional factors for the evaluation of neuronal cell culture [20]. Different parameters were analyzed to investigate the effects of the factors applied.…”

Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM‐dependent mechanism

“…Location of forelimb motor cortex was determined stereotaxically (based on retrograde tracing), and used as a guide for microdissections (Richter and Roskams, ). Cultures enriched in YFP‐expressing CST neurons were prepared as previously described, where the dissected forelimb motor cortex from 4–5 P8 homozygote Thy1‐YFP16JRS were subjected to papain (200 U; Sigma) digestion in the presence of 2 mM cysteine in dissociation medium containing 98 mM Na 2 SO 4 , 30 mM K 2 SO 4 , 5.8 mM MgCl 2 , 0.25 mM CaCl 2 , 1 mM HEPES, 20 mM D‐Glucose, and 0.125 mM NaOH, for two times 20 min at 37°C.…”