ABSTRACT
The molecular basis of axonal regeneration of central nervous system (CNS) neurons remains to be fully elucidated. In part, this is due to the difficulty in maintaining CNS neurons in vitro. Here, we show that dissociated neurons from the cerebral cortex and hippocampus of adult mice may be maintained in culture for up to 9 days in defined medium without added growth factors. Outgrowth of neurites including axons was observed from both CNS sources and was significantly greater on plasma fibronectin than on other substrata such as laminin and merosin. Neurite outgrowth on fibronectin appears to be mediated by α5ß1 integrin since a recombinant fibronectin fragment containing binding sites for this receptor was as effective as intact fibronectin in supporting neurite outgrowth. Conversely, function-blocking antibodies to α5 and ß1 integrin sub-units inhibited neurite outgrowth on intact fibronectin. These results suggest that the axonal regeneration seen in in vivo studies using fibronectin-based matrices is due to the molecule itself and not a consequence of secondary events such as cellular infiltration. They also indicate the domains of fibronectin that may be responsible for eliciting this response.
Subject(s)
Axons/drug effects , Cerebral Cortex/drug effects , Fibronectins/pharmacology , Hippocampus/drug effects , Nerve Regeneration/drug effects , Neurites/drug effects , Neurons/drug effects , Animals , Axons/physiology , Cell Adhesion/drug effects , Cell Count , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/physiology , Culture Media , Hippocampus/cytology , Hippocampus/physiology , Laminin/pharmacology , Mice , Nerve Regeneration/physiology , Neurites/physiology , Neurons/cytology , Neurons/physiologyABSTRACT
Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%). Neuronal GFP expression was observed within 18 h and continued for up to 14 days. This compares with values up to 60% of RGCs expressing GFP following infection with an HSV-1 vector. Adult rat dorsal root ganglion (DRG) neurons were also successfully transfected. Thus, in summary, Nucleofection provides the possibility for a fast and efficient method for cDNA delivery and study of gene function in adult mammalian neurons.
Subject(s)
DNA, Complementary/pharmacology , Electroporation/methods , Retinal Ganglion Cells/physiology , Transfection/methods , Age Factors , Animals , Cell Culture Techniques/methods , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cells, Cultured , DNA, Complementary/genetics , Electroporation/instrumentation , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiology , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Herpesvirus 1, Human/genetics , Rats , Rats, Wistar , Reaction Time/drug effects , Reaction Time/genetics , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/drug effects , Time Factors , Transfection/instrumentationABSTRACT
The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors. These findings indicate that developing limb buds in Xenopus release diffusible factors other than neurotrophins, able to attract growth of sensory and motor axons over long distances.
Subject(s)
Axons/physiology , Cell Movement/physiology , Limb Buds/innervation , Xenopus laevis/embryology , Ambystoma mexicanum/embryology , Animals , DNA Primers , Diffusion , Immunohistochemistry , In Vitro Techniques , Nerve Growth Factors/physiology , Reverse Transcriptase Polymerase Chain Reaction , Xenopus laevis/anatomy & histologyABSTRACT
Conditioning lesions of peripheral nerves improve axonal regeneration after injury and involve changes in expression of proteins required for axonal growth. Integrin alpha7beta1 expression in motor and sensory neurons increases following nerve lesions and motor axon regeneration is impaired in alpha7 integrin KO mice (J. Neurosci. 20, 1822-1830). To investigate the role of alpha7beta1 integrin in sensory axon regeneration, dorsal root ganglia of adult mice were cultured in gels of laminin-rich extracellular matrix (Matrigel) or collagen. Normal dorsal root ganglia in Matrigel or collagen supplemented with laminin showed spontaneous axonal outgrowth, which was greatly increased in conditioned preparations, but only in the presence of laminin. Conditioned dorsal root ganglia from normal mice cultured with a blocking antibody to beta1 integrin and from alpha7 integrin KO mice showed reduced axonal growth in both Matrigel- and laminin-supplemented collagen gels. Enhanced axonal regeneration after conditioning lesions therefore involves increased responsiveness to laminin and integrin alpha7beta1 expression.
Subject(s)
Axons/physiology , Integrins/metabolism , Nerve Regeneration/physiology , Neurons, Afferent/physiology , Animals , Antibodies/pharmacology , Axons/drug effects , Biocompatible Materials/pharmacology , Collagen/pharmacology , Drug Combinations , Female , Integrins/genetics , Integrins/immunology , Laminin/pharmacology , Male , Mice , Mice, Inbred Strains , Mice, Knockout , Neurons, Afferent/ultrastructure , Proteoglycans/pharmacologyABSTRACT
Bradykinin B2 receptor mRNA was detected at low levels, both by RT-PCR and by in situ hybridization, in freshly isolated dorsal root ganglia (DRG) and in ganglia cultured in the absence of neurotrophic factors, but was strongly upregulated by culture in the presence of nerve growth factor (NGF). The effect of NGF is mediated via TrkA receptors. The related neurotrophins, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, were ineffective in upregulating B2 mRNA, but a small upregulation was seen with the unrelated neurotrophin glial cell line-derived neurotrophic factor (GDNF). Surface membrane B2 receptor expression, detected by immunofluorescence using a B2-specific antibody, was low in outgrowing axons cultured in the absence of neurotrophic factors, but was elevated by addition of NGF or GDNF. Conditioned media prepared by incubating injured nerve, skin, or muscle had a similar effect to NGF in upregulating B2 mRNA and protein expression, and the activity was largely removed by neutralization of NGF in the conditioned medium with an anti-NGF antibody. After nerve crush injury in vivo an enhancement in B2 mRNA expression was seen, peaking after 7 days and returning to precrush levels after 14 days. In all conditions tested, the proportion of neurons expressing B2 mRNA remained the same at around 23% of small neurons, suggesting that upregulation only occurs in the B2-positive neurons. These experiments show that NGF, and to a lesser extent GDNF, upregulates the expression of bradykinin B2 mRNA and B2 receptor protein in the surface membrane of DRG neurons and that NGF is an important factor responsible for upregulating bradykinin B2 receptor expression after nerve crush injury in vivo.
