Neuron-glial interactions are important in development of the nervous system and pathogenesis of ... more Neuron-glial interactions are important in development of the nervous system and pathogenesis of disease. Primary cell cultures prepared from nervous tissue are often used to study the properties of individual cell types and how they interact with each other. Isolation of pure populations of cells and their culture is challenging, particularly from murine spinal cord. The purpose of this study was to optimize various protocols to achieve efficient, parallel isolation and purification of primary motor neurons, microglia and astrocytes from the same mouse embryonic spinal cord sample. Following dissociation of E12 embryonic spinal cords, motor neurons were isolated at 97% purity by a single step centrifugation of the cell suspension through multiple discontinuous density gradients of NycoPrep. The residual mixed cell pellet was resuspended and cultured for 2 weeks. Mixed cultures were then shaken to release microglia, which were then harvested from the medium and subjected to another round of differential adhesion to achieve 99% purity. The astrocytes remaining in the mixed cultures were culled to 98% purity by treatment with leucine methyl ester and a subsequent vigorous shaking step to remove any remaining microglia and neurons. Furthermore, no cross contamination was observed in the glial cultures. This technique provides a simple, convenient, and reliable method of obtaining highly purified preparations of motor neurons, microglia and astrocytes from embryonic spinal cord for the study of spinal cord cell biology and motor neuron diseases.
Primary motor neurons are difficult to study in conventional culture systems because of their sho... more Primary motor neurons are difficult to study in conventional culture systems because of their short-term survival without trophic support from glia. In addition, axonal migration on a two-dimensional Petri dish does not reflect the three-dimensional (3D) environment in vivo. A unique in vitro 3D model of motor nerve regeneration was developed to study motor neuron axonal migration and myelination. Mouse spinal cord motor neurons were seeded on a collagen sponge populated with Schwann cells and fibroblasts. This fibroblast-populated sponge was intended to mimic the connective tissue through which motor axons have to elongate in vivo. Addition of conventional neurotrophic supplements was not required for motor neuron survival but was necessary to promote deep neurite outgrowth, as assessed by immunostaining of neurofilament M. A vigorous neurite elongation was detected inside the sponge after only 14 days of neuron culture, reaching more than 850 microm. The model also allowed the maturation of motor fibers as one-third of them were positive for neurofilament H. Neurites growing in the sponge were subject to myelination when Schwann cells were present, as shown by myelin basic protein immunostaining and electron microscopy. We demonstrated in this model the spontaneous formation of numerous thick myelin sheaths surrounding motor fibers after long-term culture (28 days). Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on motor neurite outgrowth in vitro and also for the study of myelination and pathogenesis of motor neuron diseases.
The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Maj... more The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Major advances in our understanding of the autonomic and sensory transmission and function include the recognition of the phenotypic expression of a variety of transmitters and modulators that often coexist in individual neurons, the concept of co-transmission and chemical coding, the evidence for local effector functions of primary afferent nerves, and the discovery of plasticity of both the autonomic and the sensory nervous system during development, aging, diseases states, and inflammation. Co-transmission or plurichemical transmission, which indicates the release of more than one chemical messenger from the same neuron, enables autonomic and sensory neurons to exert a fine and highly regulated control of various functions such as circulation and immune response. The concept of chemical coding, in which the combination of transmitters/modulators is established, allows the identification of functional classes of neurons with their projections and targets. In addition to transmitters and modulators, autonomic and sensory neurons express multiple receptors, including G-proteincoupled and ion-gated receptors, further supporting the complexity of autonomic and sensory transmission and function. Autonomic neurons regulate the internal environment and maintain multiple homeostatic functions, and sensory neurons act as receptive structures that activate their targets in response to stimulation but also exert effector functions including the control of blood flow and vascular permeability, maintenance of mineralized tissue, and regulation of gene expression.
The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Maj... more The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Major advances in our understanding of the autonomic and sensory transmission and function include the recognition of the phenotypic expression of a variety of transmitters and modulators that often coexist in individual neurons, the concept of co-transmission and chemical coding, the evidence for local effector functions of primary afferent nerves, and the discovery of plasticity of both the autonomic and the sensory nervous system during development, aging, diseases states, and inflammation. Co-transmission or plurichemical transmission, which indicates the release of more than one chemical messenger from the same neuron, enables autonomic and sensory neurons to exert a fine and highly regulated control of various functions such as circulation and immune response. The concept of chemical coding, in which the combination of transmitters/modulators is established, allows the identification of functional classes of neurons with their projections and targets. In addition to transmitters and modulators, autonomic and sensory neurons express multiple receptors, including G-proteincoupled and ion-gated receptors, further supporting the complexity of autonomic and sensory transmission and function. Autonomic neurons regulate the internal environment and maintain multiple homeostatic functions, and sensory neurons act as receptive structures that activate their targets in response to stimulation but also exert effector functions including the control of blood flow and vascular permeability, maintenance of mineralized tissue, and regulation of gene expression.
Neuron-glial interactions are important in development of the nervous system and pathogenesis of ... more Neuron-glial interactions are important in development of the nervous system and pathogenesis of disease. Primary cell cultures prepared from nervous tissue are often used to study the properties of individual cell types and how they interact with each other. Isolation of pure populations of cells and their culture is challenging, particularly from murine spinal cord. The purpose of this study was to optimize various protocols to achieve efficient, parallel isolation and purification of primary motor neurons, microglia and astrocytes from the same mouse embryonic spinal cord sample. Following dissociation of E12 embryonic spinal cords, motor neurons were isolated at 97% purity by a single step centrifugation of the cell suspension through multiple discontinuous density gradients of NycoPrep. The residual mixed cell pellet was resuspended and cultured for 2 weeks. Mixed cultures were then shaken to release microglia, which were then harvested from the medium and subjected to another round of differential adhesion to achieve 99% purity. The astrocytes remaining in the mixed cultures were culled to 98% purity by treatment with leucine methyl ester and a subsequent vigorous shaking step to remove any remaining microglia and neurons. Furthermore, no cross contamination was observed in the glial cultures. This technique provides a simple, convenient, and reliable method of obtaining highly purified preparations of motor neurons, microglia and astrocytes from embryonic spinal cord for the study of spinal cord cell biology and motor neuron diseases.
Primary motor neurons are difficult to study in conventional culture systems because of their sho... more Primary motor neurons are difficult to study in conventional culture systems because of their short-term survival without trophic support from glia. In addition, axonal migration on a two-dimensional Petri dish does not reflect the three-dimensional (3D) environment in vivo. A unique in vitro 3D model of motor nerve regeneration was developed to study motor neuron axonal migration and myelination. Mouse spinal cord motor neurons were seeded on a collagen sponge populated with Schwann cells and fibroblasts. This fibroblast-populated sponge was intended to mimic the connective tissue through which motor axons have to elongate in vivo. Addition of conventional neurotrophic supplements was not required for motor neuron survival but was necessary to promote deep neurite outgrowth, as assessed by immunostaining of neurofilament M. A vigorous neurite elongation was detected inside the sponge after only 14 days of neuron culture, reaching more than 850 microm. The model also allowed the maturation of motor fibers as one-third of them were positive for neurofilament H. Neurites growing in the sponge were subject to myelination when Schwann cells were present, as shown by myelin basic protein immunostaining and electron microscopy. We demonstrated in this model the spontaneous formation of numerous thick myelin sheaths surrounding motor fibers after long-term culture (28 days). Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on motor neurite outgrowth in vitro and also for the study of myelination and pathogenesis of motor neuron diseases.
The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Maj... more The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Major advances in our understanding of the autonomic and sensory transmission and function include the recognition of the phenotypic expression of a variety of transmitters and modulators that often coexist in individual neurons, the concept of co-transmission and chemical coding, the evidence for local effector functions of primary afferent nerves, and the discovery of plasticity of both the autonomic and the sensory nervous system during development, aging, diseases states, and inflammation. Co-transmission or plurichemical transmission, which indicates the release of more than one chemical messenger from the same neuron, enables autonomic and sensory neurons to exert a fine and highly regulated control of various functions such as circulation and immune response. The concept of chemical coding, in which the combination of transmitters/modulators is established, allows the identification of functional classes of neurons with their projections and targets. In addition to transmitters and modulators, autonomic and sensory neurons express multiple receptors, including G-proteincoupled and ion-gated receptors, further supporting the complexity of autonomic and sensory transmission and function. Autonomic neurons regulate the internal environment and maintain multiple homeostatic functions, and sensory neurons act as receptive structures that activate their targets in response to stimulation but also exert effector functions including the control of blood flow and vascular permeability, maintenance of mineralized tissue, and regulation of gene expression.
The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Maj... more The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Major advances in our understanding of the autonomic and sensory transmission and function include the recognition of the phenotypic expression of a variety of transmitters and modulators that often coexist in individual neurons, the concept of co-transmission and chemical coding, the evidence for local effector functions of primary afferent nerves, and the discovery of plasticity of both the autonomic and the sensory nervous system during development, aging, diseases states, and inflammation. Co-transmission or plurichemical transmission, which indicates the release of more than one chemical messenger from the same neuron, enables autonomic and sensory neurons to exert a fine and highly regulated control of various functions such as circulation and immune response. The concept of chemical coding, in which the combination of transmitters/modulators is established, allows the identification of functional classes of neurons with their projections and targets. In addition to transmitters and modulators, autonomic and sensory neurons express multiple receptors, including G-proteincoupled and ion-gated receptors, further supporting the complexity of autonomic and sensory transmission and function. Autonomic neurons regulate the internal environment and maintain multiple homeostatic functions, and sensory neurons act as receptive structures that activate their targets in response to stimulation but also exert effector functions including the control of blood flow and vascular permeability, maintenance of mineralized tissue, and regulation of gene expression.
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