Embryonic Dorsal Root Ganglion Cells Research Articles

Innervation of Meissner's corpuscles and Merkel -cells by transplantation of embryonic dorsal root ganglion cells after peripheral nerve section in rats.

Transplantation of embryonic motor neurons has been shown to improve motor neuron survival and innervation of neuromuscular junctions in peripheral nerves. However, there have been no reports regarding transplantation of sensory neurons and innervation of sensory receptors. Therefore, we hypothesized that the transplantation of embryonic sensory neurons may improve sensory neurons in the skin and innervate Merkel cells and Meissner's corpuscles. We obtained sensory neurons from dorsal root ganglia of 14-day rat embryos. We generated a rat model of Wallerian-degeneration by performing sciatic nerve transection and waiting for one week after. Six months after cell transplantation, we performed histological and electrophysiological examinations in naïve control, surgical control, and cell transplantation groups. The number of nerve fibers in the papillary dermis and epidermal-dermal interface was significantly greater in the cell transplantation than in the surgical control group. The percent of Merkel cells with nerve terminals, as well as the average number of Meissner corpuscles with nerve terminals, were higher in the cell transplantation than in the surgical control group, but differences were not significant between the two groups. Moreover, the amplitude and latency of sensory conduction velocity were evoked in rats of the cell transplantation group. We demonstrated that the transplantation of embryonic dorsal root ganglion cells improved sensory nerve fiber number and innervation of Merkel cells and Meissner's corpuscles in peripheral nerves.

Muscle spindle reinnervation using transplanted embryonic dorsal root ganglion cells after peripheral nerve transection in rats

ObjectivesMuscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles.Materials and methodsWe collected sensory neurons from dorsal root ganglions of 14‐day‐old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups.ResultsOur results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D‐28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group.ConclusionsCell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle.

Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons

IgLONs are members of the immunoglobulin superfamily of cell adhesion proteins implicated in the process of neuronal outgrowth, cell adhesion and subdomain target recognition. IgLONs form homophilic and heterophilic complexes on the cell surface that repress or promote growth depending on the neuronal population, the developmental stage and surface repertoire of IgLON family members. In the present study, we identified a metalloproteinase-dependent mechanism necessary to promote growth in embryonic dorsal root ganglion cells (DRGs). Treatment of embryonic DRG neurons with pan-metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-3, or an inhibitor of ADAM Metallopeptidase Domain 10 (ADAM10) reduces outgrowth from DRG neurons indicating that metalloproteinase activity is important for outgrowth. The IgLON family members Neurotrimin (NTM) and Limbic System-Associated Membrane Protein (LSAMP) were identified as ADAM10 substrates that are shed from the cell surface of DRG neurons. Overexpression of LSAMP and NTM suppresses outgrowth from DRG neurons. Furthermore, LSAMP loss of function decreases the outgrowth sensitivity to an ADAM10 inhibitor. Together our findings support a role for ADAM-dependent shedding of cell surface LSAMP in promoting outgrowth from DRG neurons.

Coculture of Schwann Cells and Neuronal Cells for Myelination in Rat

For in vitro myelination system, Schwann cells and neuronal cells of rat were cocultured. Schwann cells and neuronal cells, respectively, were obtained from dorsal root ganglion of rat embryos (E15). This method includes four steps: first step of suspension of the embryonic dorsal root ganglion cells, second step of addition of anti-mitotic cocktail, third step of purification of dorsal root cells, and fourth step of addition of Schwann cells to dorsal root ganglion cells. We made a highly purified population of myelination in a short period through this procedure and identified myelination basic protein using antibody of myelination basic protein.

Research Highlights: Highlights From The Latest Articles in Nanomedicine

Nanomedicine (2014) 9(5), 573–576 ISSN 1743-5889 Aligned nanofibers give sense of direction to cells Evaluation of: Berns EJ, Sur S, Liuliu P et al. Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels. Biomaterials 35(1), 185–195 (2014). The dual nature of peptide amphiphiles (PAs), composed of an aliphatic hydrophobic part combined with a peptide section, has been exploited in the generation of nano­ structured gels. Previous work reported by Zhang et al. [1] already demonstrated the possibility of using PAs to generate aligned nanofibers through a thermal annealing pro­ cess. In the present work, Berns et al. demon­ strate that the bioactivity of this system can be further enhanced using co­assembly of a structuring PA with the capacity to form self­assembled nanofibers and functional PAs that present bioactive peptide epitopes (i.e., RGDS and IKVAV). The injection of the thermally annealed PA solutions into a calcium chloride solu­ tion generates noodle­like macroscopic struc­ tures composed of aligned nanofibers. A series of tests using different cells show that these nano­structured materials promote the regeneration of neural tissue; P19 cells cultured on these scaffolds present a direc­ tional neurite outgrowth aligned with the nanofibers. Moreover, neurons cultured on the aligned scaffolds form synaptic connec­ tions and propagate electrical signals. This is an indication that the scaffold allows trans­ fer of information by the neurons in a direc­ tional manner. Moreover, the authors dem­ onstrate that embryonic dorsal root ganglia cells migrate following the direction of the nanofibers. The results presented by Berns et al. demon strate the advantages that the co­ assembly can offer. Undoubtedly, the described nano­aligned scaffolds (with and without the presence of bioactive epitopes) fill a niche in the engineering of biomaterials for regeneration of tissues that require cell alignment or directional cell migration, as well as directional cell–cell communication (e.g., neural tissues, tendon, muscle, and so on).

Research Highlights: Highlights From The Latest Articles in Nanomedicine

Nanomedicine (2014) 9(5), 573–576 ISSN 1743-5889 Aligned nanofibers give sense of direction to cells Evaluation of: Berns EJ, Sur S, Liuliu P et al. Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels. Biomaterials 35(1), 185–195 (2014). The dual nature of peptide amphiphiles (PAs), composed of an aliphatic hydrophobic part combined with a peptide section, has been exploited in the generation of nano­ structured gels. Previous work reported by Zhang et al. [1] already demonstrated the possibility of using PAs to generate aligned nanofibers through a thermal annealing pro­ cess. In the present work, Berns et al. demon­ strate that the bioactivity of this system can be further enhanced using co­assembly of a structuring PA with the capacity to form self­assembled nanofibers and functional PAs that present bioactive peptide epitopes (i.e., RGDS and IKVAV). The injection of the thermally annealed PA solutions into a calcium chloride solu­ tion generates noodle­like macroscopic struc­ tures composed of aligned nanofibers. A series of tests using different cells show that these nano­structured materials promote the regeneration of neural tissue; P19 cells cultured on these scaffolds present a direc­ tional neurite outgrowth aligned with the nanofibers. Moreover, neurons cultured on the aligned scaffolds form synaptic connec­ tions and propagate electrical signals. This is an indication that the scaffold allows trans­ fer of information by the neurons in a direc­ tional manner. Moreover, the authors dem­ onstrate that embryonic dorsal root ganglia cells migrate following the direction of the nanofibers. The results presented by Berns et al. demon strate the advantages that the co­ assembly can offer. Undoubtedly, the described nano­aligned scaffolds (with and without the presence of bioactive epitopes) fill a niche in the engineering of biomaterials for regeneration of tissues that require cell alignment or directional cell migration, as well as directional cell–cell communication (e.g., neural tissues, tendon, muscle, and so on).

Induction of neuronal and myelin-related gene expression by IL-6-receptor/IL-6: A study on embryonic dorsal root ganglia cells and isolated Schwann cells

The conditional knockdown of the Interleukin-6 (IL-6) family signal transducer (gp130) causes peripheral nerve demyelination and degeneration. In the present work, we investigated the effect of gp130 signaling on peripheral nerves and Schwann cells (SC). We stimulated gp130 signaling with IL6RIL6, a fusion molecule of IL-6 and IL-6R, in rat embryonic day 14 dorsal root ganglia (DRG) cell cultures. In neurons, IL6RIL6 strongly increased the axonal network. In SC, IL6RIL6 favored the appearance of elongated more mature cells versus stellar shaped cells. Gene expression profiling showed an increased expression of neuronal and glial-specific genes. mRNAs related to SC function, including myelin-specific genes, were increased by IL6RIL6 treatment of DRG cells, or of purified SCs isolated from rat sciatic nerve. In IL6RIL6-treated cells, immunostaining showed a strong nuclear signal for Krox-20, a transcription factor essential for differentiation of the SC lineage. On the contrary, we observed that IL6RIL6 inhibited the genes related to TGF-beta family as well as the production of smooth muscle actin.

A cell therapy approach to substitute neural elements in the inner ear

Three different donor tissues were tested for their capacity to survive, integrate and differentiate in the adult inner ear. Surviving embryonic dorsal root ganglion cells were found within the spiral ganglion neuron region and along the auditory nerve fibers. In the presence of exogenous nerve growth factor (NGF), the dorsal root ganglion cells formed extensive growth of neurites that seemed to contact the host neurons. Adult neural stem cells survived relative poorly in the inner ear whereas embryonic stem cells showed a somewhat greater capacity for survival and integration. Overall, the survival rate of implanted tissue was quite low in the cochlea. It is concluded that an inner ear cell therapy approach based on the implantation of exogenous cells will require that important survival factors are identified and supplied. In addition, it is possible that the physical properties of the cochlea, e.g., fluid-filled compartments and very limited space for cell proliferation, are unfavorable, at least in the normal cochlea.

Functional connections are established in the deafferented rat spinal cord by peripherally transplanted human embryonic sensory neurons.

Functionally useful repair of the mature spinal cord following injury requires axon growth and the re-establishment of specific synaptic connections. We have shown previously that axons from peripherally grafted human embryonic dorsal root ganglion cells grow for long distances in adult host rat dorsal roots, traverse the interface between the peripheral and central nervous system, and enter the spinal cord to arborize in the dorsal horn. Here we show that these transplants mediate synaptic activity in the host spinal cord. Dorsal root ganglia from human embryonic donors were transplanted in place of native adult rat ganglia. Two to three months after transplantation the recipient rats were examined anatomically and physiologically. Human fibres labelled with a human-specific axon marker were distributed in superficial as well as deep laminae of the recipient rat spinal cord. About 36% of the grafted neurons were double labelled following injections of the fluorescent tracers MiniRuby into the sciatic and Fluoro-Gold into the lower lumbar spinal cord, indicating that some of the grafted neurons had grown processes into the spinal cord as well as towards the denervated peripheral targets. Electrophysiological recordings demonstrated that the transplanted human dorsal roots conducted impulses that evoked postsynaptic activity in dorsal horn neurons and polysynaptic reflexes in ipsilateral ventral roots. The time course of the synaptic activation indicated that the human fibres were non-myelinated or thinly myelinated. Our findings show that growing human sensory nerve fibres which enter the adult deafferentated rat spinal cord become anatomically and physiologically integrated into functional spinal circuits.

Overexpression of activated neu/erbB2 initiates immortalization and malignant transformation of immature Schwann cells in vitro.

The neu/erbB2 protooncogene is overexpressed in numerous human cancers and is mutationally activated in N-ethyl-N-nitrosourea (ENU)-induced rodent tumors of the Schwann cell lineage. We investigated whether expression of activated neu in Schwann cells is sufficient to initiate their immortalization and transformation. Clones of embryonic dorsal root ganglia cells infected with a retrovirus bearing activated neu (NID cells) were selected based on their expression of Schwann cell-specific markers. Compared to embryonic Schwann cells infected with a virus encoding empty vector, we found that NID cells have altered shapes and disorganized cytoskeletons, grow in the absence of growth factors required for normal Schwann cell survival and proliferation, and can be repeatedly passaged. Furthermore, NID cells are invasive in an in vitro matrix invasion assay and form metastatic tumors when injected into syngeneic animals. The neu-induced growth and invasive phenotypes could be reversed by drugs that inhibit Ras and Src activity. Interestingly, later stage Schwann cells infected with activated neu failed to become immortalized. These findings indicate that constitutive activation of erbB2 is sufficient to initiate the immortalization and transformation of immature Schwann cells, and support the notion that Schwann cells have particular developmental stages during which they are susceptible to immortalizing and transforming events.

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone.

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5-8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.

Action potential propagation through embryonic dorsal root ganglion cells in culture. II. Decrease of conduction reliability during repetitive stimulation.

1. The reliability of the propagation of action potentials (AP) through dorsal root ganglion (DRG) cells in embryonic slice cultures was investigated during repetitive stimulation at 1-20 Hz. Membrane potentials of DRG cells were recorded intracellularly while the axons were stimulated by an extracellular electrode. 2. In analogy to the double-pulse experiments reported previously, either one or two types of propagation failures were recorded during repetitive stimulation, depending on the cell morphology. In contrast to the double-pulse experiments, the failures appeared at longer interpulse intervals and usually only after several tens of stimuli with reliable propagation. 3. In the period with reliable propagation before the failures, a decrease in the conduction velocity and in the amplitude of the afterhyperpolarization (AHP), an increase in the total membrane conductance, and the disappearance of the action potential "shoulder" were observed. 4. The reliability of conduction during repetitive stimulation was improved by lowering the extracellular calcium concentration or by replacing the extracellular calcium by strontium. The reliability of conduction decreased by the application of cadmium, a calcium channel blocker, 4-amino pyridine, a fast potassium channel blocker, or apamin or muscarine, the blockers of calcium-dependent potassium channels. The reliability of conduction was not effected by blocking the sodium potassium pump with ouabain or by replacing extracellular sodium with lithium. 5. In the period with reliable propagation cadmium, apamin, and muscarine reduced the amplitude of the AHP. The shoulder of the action potential was more pronounced and not sensitive to repetitive stimulation when extracellular calcium was replaced by strontium. It disappeared when cadmium was applied. 6. In DRG somata changes of the intracellular Ca2+ concentration were monitored by measuring the fluorescence of the Ca2+ indicator Fluo-3 with a laser-scanning confocal microscope. During repetitive stimulation, an accumulation of intracellular calcium occurred that recovered very slowly (tens of seconds) after the AP trains. 7. Computer model simulations performed in analogy to the experimental protocols produced conduction failures during repetitive stimulation only when the calcium currents during the APs were reduced. 8. From these findings it is concluded that conduction failures during repetitive stimulation are dependent on an accumulation of intracellular calcium leading to an inactivation of calcium currents, combined with small contributions of an accumulation of extracellular potassium and a summation of slow potassium conductances.

Expression of calcium-binding proteins in the neurotrophin-3-dependent subpopulation of rat embryonic dorsal root ganglion cells in culture

In this study we have examined the calcium-binding protein expression in rat embryonic (E16) dorsal root ganglia (DRG) neurons in vitro in the presence of neurotrophin-3 (NT-3). A comparison was made with the expression of calcium-binding proteins in DRG subpopulations that depended in vitro on nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF). Our results show that NT-3 promotes the survival of a DRG subpopulation of which over 75% expresses parvalbumin (PV). The majority of these PV-positive NT-3-dependent DRG neurons were large ‘type A’ neurons. Expression of calbindin-D28k (CaBP) and calretinin (Calr) in the NT-3-dependent DRG population was seen in smaller fractions (between 12 and 17%) of the surviving DRG neurons and in both type A and B neurons. The preferential expression of PV in NT-3-dependent type A neurons is unique in comparison to the expression of PV and the other calcium-binding proteins in DRG neurons surviving in vitro in the presence of NGF or BDNF.

Action potential propagation through embryonic dorsal root ganglion cells in culture. I. Influence of the cell morphology on propagation properties.

1. In this and the companion paper the reliability of action potential (AP) propagation through dorsal root ganglion (DRG) cells was investigated. Experimental data were collected from DRG cells of embryonic rat slice cultures of the spinal cord. A field stimulation electrode was used to elicit an AP in the axon. The propagated AP or, in case of conduction block, its electronic residue (ER), was measured intracellularly in the soma of the DRG cell. 2. The morphological and electrophysiological data combined with published data from voltage-clamp studies were taken to implement a compartmental computer model, which allows a precise description of the propagating AP and the channel kinetics at any point along the axon. 3. The safety factor for conduction was found to be low. Thus failures of AP invasion of the DRG cell soma could occur at sites of impedance mismatch when a hyperpolarizing current was applied, a second stimulus felt into the relative refractory period of the first, or when the axon was repetitively stimulated. 4. The ERs of the failed APs had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. These sites of low safety factor were found to be the branch point in the unipolar DRG cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. 5. A systematic comparison of bipolar and unipolar DRG cells showed that the AP conduction through the latter is more reliable. For large cell bodies, the unipolar configuration is needed for save conduction. 6. Conduction through unipolar DRG cells is faster than through bipolar cells because the electrical load of the soma is masked by the high-resistive stem piece. The length of this stem piece is correlated inversely to the delay caused at the branch point, as the electrical load of the soma is more efficiently masked by a long stem piece.

Large unit conductance voltage chloride channels are expressed in mouse neural crest cells and embryonic dorsal root ganglion cells

The early expression of voltage-activated chloride channels of large unitary conductance (450 pS in symmetrical 140 mM KCl) was demonstrated using patch-clamp techniques in two preparations: (i) neural crest cells isolated from 9-day-old (E9) mouse embryos and (ii) acutely isolated dorsal root ganglion cells isolated from E12 mouse embryos. Properties of these ionic channels have been analyzed using single channel recordings and the group mean of these single channels.

Herpes simplex virus infection of the human sensory neuron. An electron microscopy study.

Herpes simplex virus (HSV) type 1 was used to infect cultures of human embryonic dorsal root ganglion cells. Infected cultured were studied by electron microscopy. Viral nucleocapsids were observed to be internalized into neuronal cells bodies and neuritic extensions by fusion of the viral envelope and the plasma membrane. No signs of internalization by endocytosis were noted. Nucleocapsids were transported in neurites and were within 2 hrs postinfection found located near the microtubules and close to the nuclear pores in the perikaryon. A primary envelopment of nucleocapsids occurred at the inner lamina of the nuclear membrane and virions appeared between the two laminae. Presence of non-enveloped nucleocapsids outside the nuclear membrane and in close contact with the endoplasmic reticulum suggested that nucleocapsids could pass to the cytoplasmic side probably by de-envelopment at the outer nuclear membrane. A secondary envelopment occurred at the endoplasmic reticulum where the virions also became enclosed in transport vesicles. Enveloped virus appearing in the cytoplasm of neurons and neuritic extensions was always found only inside these transport vesicles. During their passage through the cytoplasm the virion-transport vesicle complexes were surrounded by smaller lysosome-like vesicles possibly derived from the Golgi apparatus. Fusion reactions between vesicles with virions and the smaller vesicles seemed to occur. We discuss if in this way the virion-transport vesicle complexes might be provided with glycosyl transferases and substrates necessary for maturation and completion of glycosylation of the viral envelope glycoproteins. The transport vesicles seemed essential for egress of virions from the infected cell by releasing virus when fusing with the plasma membrane.

Rabies virus infection of cultured rat sensory neurons

The axonal transport of rabies virus (challenge virus strain of fixed virus) was studied in differentiated rat embryonic dorsal root ganglion cells. In addition, we observed the attachment of rabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infection and manipulation of neuronal extensions without exposing the neural soma to the virus. The cultures consisted of 60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated high binding affinity to unmyelinated neurites, as suggested by assays of virus adsorption and immunofluorescence studies. The rate of axoplasmic transport of virus was 12 to 24 mm/day, including the time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, and colchicine, none of which negatively affected the production of virus in cells once the infection was established. It was concluded that, for the retrograde transfer of rabies virus by neurites from the periphery to the neuronal soma, the integrity of tubulin- and actin-containing structures is essential. The rat sensory neurons were characterized as permissive, moderately susceptible, but low producers of rabies virus. These neurons were capable of harboring rabies virus for long periods of time and able to release virus into the culture medium without showing any morphological alterations. The involvement of sensory neurons in rabies virus pathogenesis, both in viral transport and as a site for persistent viral infection, is discussed.

Developmental capacities of avian embryonic dorsal root ganglion cells: neuropeptides and tyrosine hydroxylase in dissociated cell cultures

Dorsal root ganglia (DRG) from quail embryos of 10–15 days of incubation (E10–15) contain a subpopulation of cells, distinct from postmitotic neurons, that can, under suitable conditions of culture in vitro, differentiate into neuron-like cells that display a variety of adrenergic properties, including tyrosine hydroxylase (TH) immunoreactivity (Xue et al., Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8800–8804). The present study was undertaken to determine whether other markers typical of autonomic sympathetic nerve cells are also expressed in the same system. Cells immunoreactive for vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were found to differentiate continually from non-dividing precursors in all cultures of dissociated E10 quail DRG grown in the presence of chick embryo extract. Whereas VIP was already present (in a minute number of cells) in DRG in situ, NPY could not be detected before 3 days of culture, when it appeared simultaneously with TH. Double immunostaining experiments showed that most VIP-positive cells and about half the NPY-positive cells also displayed TH-immunoreactivity. On the other hand, there was no overlap between the substance P-containing neuronal population and any of the cells containing TH, NPY or VIP. These observations are pertinent to the problem of the segregation of autonomic and sensory cell lines during peripheral nervous system ontogeny.