An Experimental Study Of The Phenotype Of DRG Neurons Affected By GM1, Neurotrophins And Skeletal Muscle Cells

The neuropeptide-immunoreactive(NP-IR) and neurofilament-IR(NF-IR) neurons are two major phenotypical classes in dorsal root ganglion(DRG).NP-IR neurons are considered to be with unmyelinated or thinly myelinated nociceptive afferents which are considered to innervate skin and viscera.Neuropeptide-IR neurons synthesized and released a variety of bioactive substances,including substance P(SP).Whereas neurofilament-IR neurons typically have myelinated axons which are considered to innervate muscle spindle,involved in the stretch reflex and proprioception.The neurotransmitters of SP are located in the DRG neurons,synthesized in the cell bodies and transported to the axon terminals in the state of inflammation,stress and other stimuli.Neurofilaments are the important structural component of the neurons cytoskeleton,involved in the maintenance of normal structure and morphology.There are three neurofilament subtypes,NF-light, medium,and heavy,that is,NFL,NFM and NFH.The molecular weight was 68 kDa,160 kDa and 200 kDa,respectively.NF-200 is the main component of the cells skeleton structure.In DRG neurons,it is expressed during development.It is an important indicator for the identification of NF-IR neuronal phenotype.Monosialoganglioside(GMI) plays a nutritional role in neuronal development, differentiation,and survival in the condition of physiological and pathological.Both in vivo and in vitro experiments showed that,GM1 can play a neurotrophic factor-like role in neurons.In vitro,the differentiation of neurons is accompanied by the biosynthesis of GM1 and the content increased of membrane GM1.The motor neurons and skeletal muscle fibers they innervate are strongly dependent on each other.Important communication between both tissues is mediated through the neuromuscular junction.However,release and reception of various factors at other parts of both tissues must also be considered as means of mutual influences.Exchange of neurotrophins and other molecules is likely to be an important source of nerve-muscle communication.The in vitro survival of neonatal mammalian neurofilament-positive DRG neurons requires the presence of a neurotrophic factor present in skeletal muscle extract.The experimental results show that during the development of neurons,target-derived neurotrophic factor has an important role in the survival and differentiation of the innervated neurons. Skeletal muscles(i.e.,muscle spindle) are also innervated by DRG sensory neurons which are considered as neurofilament-IR neurons.Neurotrophins(NTs) are the most profound known regulators of survival in the developing peripheral nervous system.Nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),and neurotrophin 3(NT-3) are three members of the neurotrophic factor(neurotrophin) family.Exogenous administration of these factors has protective properties for injured neurons and stimulates axonal regeneration.NGF has a key role in the survival and establishment of the phenotype of responsive primary afferent neurons during development.Some neuronal phenotypes of neuropeptides are retained and can remain sensitive to NGF regulation in aged DRG neuronal cultures.NGF regulates sensory neuron phenotype by elevated expression of ion channels and receptors contributing to pain. After the DRG neurons damaged,NGF counteracted this injury-induced reduction of neurofilament mRNA but only in neurons with high-affinity NGF receptors. BDNF plays a critical role in the development of the central and peripheral nervous systems,supports proliferation,differentiation and survival of neurons.It has been shown that BDNF is present in a subpopulation of small- to medium-sized DRG sensory neurons and is anterogradely transported in both the peripheral and central processes.The presence of BDNF is necessary for the maintenance of the DRG neurons in vivo.In vitro studies suggest that survival of many populations of cranial sensory neurons initially depends on BDNF.NT-3 is also a key member of neurotrophin family.NT-3 has an important role in survival and differentiation of neurons served as a factor of promoting cell division or inducing neuronal differentiation.NT-3 plays an important role in survival and differentiation of sensory and sympathetic neurons,sprouting of neurites,synaptic reorganization,and axonal growth.GM1,NGF,BDNF,NT-3 and target tissues are essential for the maintenance of the function of neurons.However,much less is known about the influence of GM1, NGF,BDNF,NT-3 and target muscle cells on DRG neuronal phenotypes.And also, association of target muscle cells with GM1,NGF,BDNF and NT-3 on influence of DRG neuronal phenotype remains unknown.Here we have established a single DRG neurons culture model and a neuromuscular co-culture model of DRG neurons and skeletal muscle cells to test what extent to the expression of SP and NF-200 in DRG neurons in both cell culture models.PartⅠEffects of GM1 and skeletal muscle cells on dorsal root ganglion neuronal phenotypes in vitroDRG was harvested at embryonic days 12.5(El2.5) and E19.5,and cultured without and with skeletal muscle cells(SKM).After cultured for 2 d,the cultures were divided into 8 groups.(1) E12.5 control:E12.5 DRG neurons was cultured continuously for another 4 d;(2) E19.5 control:E19.5 DRG neurons was cultured continuously for another 4 d;(3) E12.5 GM1 group:E12.5 DRG neuronal culture was exposed to GM1(final concentration,FC:10μg/ml) for another 4 d;(4) E19.5 GMI group:E19.5 DRG neuronal culture was exposed to GMI(final concentration, FC:10μg/ml) for another 4 d;(5) E12.5 SKM group:The co-culture of E12.5 DRG neurons and SKM cells was cultured for another 4 d;(6) E19.5 SKM group:The co-culture of E19.5 DRG neurons and SKM cells was cultured for another 4 d;(7) E12.5 GMI+SKM group:The co-culture of E12.5 DRG neurons and SKM cells with GMI exposure(FC:10μg/ml) for another 4 d;(8) E19.5 GM1+SKM group: The co-culture of E19.5 DRG neurons and SKM cells with GMI exposure(FC:10μg/ml) for another 4 d.All the cultured living cells were observed under inverted phase contrast microscope.The morphology of DRG neurons and the relationship between DRG neuronal terminals and SKM cells were observed by using scanning electron microscope(SEM) at 6 d of culture age.DRG cultures and neuromuscular co-cultures were processed for double immunofluorescent labeling of MAP2 and SP or NF-200 at 6 d of culture age.The results are as follows:(1) Under inverted phase contrast microscope and scanning electron microscopy,it has shown that many DRG neurons neurites crossed or distributed to the surface of the SKM cells.(2) GM1 could promote SP and NF-200 expression in the cultures of DRG harvested at E12.5(P＜0.05).GMI had not effects on SP and NF-200 expression in the cultures of DRG harvested at E19.5.(3) Target SKM could promote expression of NF-200(P＜0.05) but not SP in the cultures of DRG harvested at E12.5.Target skeletal muscle cells had not effects on SP and NF-200 expression in the cultures of DRG harvested at E19.5.(4) GM1 and target skeletal muscle cells had synergistic effects on the expression of SP and NF-200 in the cultures of DRG harvested at E12.5(P＜0.001). Target skeletal muscle cells combined with GM1 had not effects on SP and NF-200 expression in the cultures of DRG harvested at E19.5.The results indicate that the contact between DRG neurons and skeletal muscle cells was founded in the neuromuscular co-culture of DRG neurons and skeletal muscle cells.These data suggest that neurofilament-IR neurons,but not neuropeptide-IR neurons,are partially dependent on the presence of target skeletal muscle cells.In the present study,GM1 promoted SP and NF-200 expression in E12.5 DRG neuronal cultures.Target skeletal muscle cells only induced NF-200, but not SP expression in E12.5 DRG neuronal cultures.Furthermore,GM1 and target skeletal muscle cells had synergistic effects on SP and NF-200 expression in E12.5 DRG neuronal cultures.These results indicated that the cultured DRG neurons which were harvested at times before(at E12.5) sensory neurons contact peripheral targets in vivo have some plasticity in the presence of GM1.However, the DRG neurons phenotype harvested after(at E19.5) sensory neurons contact peripheral targets in vivo are not dependent on the presence of target skeletal muscle cells and GM1.These results offered new clues for a better understanding of the association ofGMl and skeletal muscle with neuronal phenotypes.PartⅡEffects of neurotrophins and skeletal muscle cells on dorsal root ganglion neuronal phenotypes in vitroDRG cell culture preparations utilized E12.5 d Wistar rats.Under aseptic conditions,DRG neurons are cultured as dissociate cells or cultured as the neuromuscular co-cultures of DRG neurons and SKM cells.After cultured for 2d, the cultures were divided into 8 groups.(1) Control:DRG neurons was cultured continuously for another 4 d;(2) NGF group:DRG neuronal culture was exposed to NGF(FC:10 ng/ml) for another 4 d;(3) BDNF group:DRG neuronal culture was exposed to BDNF(FC:10 ng/ml) for another 4 d;(4) NT-3 group:DRG neuronal culture was exposed to NT-3(FC:10 ng/ml) for another 4 d;(5) SKM group:The co-culture of DRG neurons and SKM cells was cultured for another 4 d;(6) SKM+NGF group:The co-culture of DRG neurons and SKM cells with NGF exposure(FC:10 ng/ml) for another 4 d;(7) SKM+ BDNF group:The co-culture of DRG neurons and SKM cells with BDNF exposure(FC:10 ng/ml) for another 4 d; (8) SKM+ NT-3 group:The co-culture of DRG neurons and SKM cells with NT-3 exposure(FC:10 ng/ml) for another 4 d.All the cultured living cells were observed under inverted phase contrast microscope.At 6 d of culture age,all above cultured samples were processed for detecting the expression of SP and NF-200 in DRG neurons by double immunofluorescent technique.The neuromuscular co-culture samples were processed for detecting the expression of SP and NF-200 in DRG neurons by Western blot analysis.The results are as follows:(1) NGF could promote SP(P＜0.01) and NF-200(P＜0.05) expression in the cultures of DRG harvested at E12.5 d,and NGF and target SKM cells had synergistic effects on the expression of SP and NF-200 in the cultures of DRG harvested before sensory neurons contact peripheral targets in vivo(E12.5).(2) BDNF could promote SP and NF-200 expression in the cultures of DRG harvested at E12.5 d(P＜0.05),and BDNF and target SKM cells had synergistic effects on the expression of NF-200 in the cultures of DRG harvested at E12.5.(3) NT-3 and target SKM cells could promote expression of NF-200(P＜0.05) but not SP in the cultures of DRG harvested at E12.5 d,and NT-3 and target SKM cells had synergistic effects on the expression of NF-200 in the cultures of DRG harvested at E12.5(P＜0.001).The results indicate that NGF,BDNF or NT-3 have an effect on the expression of DRG neurons phenotype in the cultures of DRG harvested before sensory neurons contact peripheral targets in vivo(E12.5).Neurothophins and target tissues have different effects on the phenotype of DRG neurons.NGF and BDNF play a key role in the NP-IR and NF-IR phenotype of DRG neurons,but NT-3 only have a role in the NF-IR phenotype of DRG neurons.NGF and SKM cells had synergistic effects on the two major phenotypicai classes in DRG.BDNF and NT-3 had synergistic effects on the NF-IR phenotype of DRG neurons in the presence of SKM cells.These datas showed that the DRG neurons phenotypic plasticity is different in different conditions during the development.The mechanism involved in these effects should be clarified by further study.These results provided a new way to find the mechanism of the changes of neuronal phenotypes affected by the NTs and target SKM cells.