The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodendrocytes in culture

A neuronal culture system has been developed that has demonstrated to induce myelin formation by added oligodendrocytes. Networks of dissociated dorsal root ganglion neurons were prepared by suppressing non-neuronal cells (i.e. fibroblasts and Schwann cells) with a continuous 2 week exposure to 10⁻⁵ M fluorodeoxyuridine in the culture medium. After drug withdrawal, neuroglial cells were introduced in optic nerve implants from 1–2 week-old rats. These added glial cells migrated extensively over the unensheathed neurities and central myelin was formed by 2 weeks after the implant addition.     

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.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

Ontogeny of enkephalin- and VIP-containing neurons in dissociated cultures of embryonic mouse spinal cord and dorsal root ganglia

The ontogeny of vasoactive intestinal polypeptide (VIP), and Met-enkephalin in primary cultures of spinal cord/dorsal root ganglia from 12-day mouse embryos was examined by radioimmunoassay and immunohistochemistry. Met-enkephalin levels rose from less than 5 to 700 pg/culture over 26 days and were half maximal by day 16-18 in culture. VIP levels rose from less than 1 to 30 pg/culture over the same period, but were already half maximal by day 9. Met-enkephalin immunoreactivity was localized in multipolar medium sized neurons while VIP immunoreactivity was visualized both in neurons with extensively branched processes and in bipolar cells some of which appeared to be dorsal root ganglion cells. Tetrodotoxin (TTX)-sensitive spontaneous release of both peptides developed in parallel with the ability to stimulate peptide release with elevated potassium. Factors affecting the ontogeny of neuropeptide expression in, and release from, spinal cord neurons can now be examined in vitro in a strictly defined neurochemical environment.     

Antioxidants are necessary for myelination of dorsal root ganglion neurons, in vitro

We have demonstrated that myelination of dorsal root ganglion (DRG) axons occurs in a fully defined, serum-free medium (B27). This implies that there may be components in B27 medium that support myelination. To determine which of the components in B27 were essential for myelination, we systematically removed components from B27 until myelination was lost. We added these components to a fully defined minimal medium (N2) that supports neuron survival but not myelination. When antioxidants were removed from B27, myelination was lost. However, the individual antioxidants did not induce myelination when added to N2 medium. Addition of ascorbic acid along with the B27 antioxidants was sufficient to induce myelination in N2 medium, which was enhanced by retinyl acetate. Removal of vitamin E from B27 caused a partial loss of myelination, and addition of vitamin E to N2 medium containing ascorbic acid induced partial myelination. Addition of serum to the B27 myelinating medium inhibited myelination completely. These results indicate that antioxidants are important for myelination, in vitro. Vitamin E may play an important role. Use of a serum-free medium may be beneficial for in vitro myelination studies because serum has unknown inhibitory effects.     

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.     

Ultrastructural effects of Guillain-Barré serum in cultures containing only rat Schwann cells and dorsal root ganglion neurons

Serum from patients with the acute form of the Guillain-Barré syndrome was applied to cultures containing only rat dorsal root ganglion neurons and Schwann cells. Serum taken from 4 of 10 patients during the first 1-3 weeks of clinical onset had previously been shown to have significant demyelinating activity in this culture system when observed at the light microscopic level. More detailed assessment made at the ultrastructural level showed that: (1) wide-spread myelin-related Schwann cell lysis occurred in concert with vesicular myelin breakdown; (2) non-myelin-related Schwann cells avidly phagocytized necrotic cell debris and fragments of compact myelin; and (3) neurites and non-myelin-related Schwann cells remained structurally undamaged. Cultures treated with convalescent phase serum from patients whose acute phase serum had cytolytic activity displayed no significant ultrastructural damage to either neurites or Schwann cells. This is the first electron microscopic study to provide direct evidence that acute Guillain-Barré serum can be cytolytic for myelin-related Schwann cells and peripheral myelin in an experimental setting free of leukocytes, lymphocytes and mononuclear phagocytes.     

Receptor-mediated uptake of labeled transferrin by embryonic chicken dorsal root ganglion neurons in culture

Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM ¹²⁵I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized ¹²⁵I-transferrin was quantified with a gamma counter. ¹²⁵I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 μM unlabeled transferrin. No competition for the uptake of ¹²⁵I-transferrin was observed in the presence of 1.25 μM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment (‘half-site’) of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment (‘half-site’) of transferrin (1.25 μM) or in the presence of concanavalin A (1.25 μM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons.     

Substance P-like immunoreactivity in neurons in dissociated cell cultures of mammalian spinal cord and dorsal root ganglia

Dissociated cell cultures prepared from fetal mouse spinal cords and dorsal root ganglia were stained for endogenous substance P using the peroxidase-antiperoxidase technique. Substance P-like immunoreactivity was localized within a small percentage of rounded or multipolar neuronal somata and in varicose processes. The substance P-positive multipolar neurons were derived from spinal cord, while the small rounded neurons were possibly of spinal cord and/or sensory ganglion origin. Large dorsal root ganglion neurons were unreactive. These results are consistent with in vivo findings and indicate the feasibility of electrophysiologic studies in culture to analyze the synaptic connections between substance P neurons and their target cells.     

Immunohistochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion neurons in culture

The addition of central nervous system (CNS) glial cells to dissociated networks of rat dorsal root ganglion neurons in tissue culture provided a useful system for the study of CNS myelin sheath formation. The CNS myelin basic proteins (BP) and proteolipid protein (PLP) were demonstrable in these cultures by immunoperoxidase techniques. Both BP and PLP were detectable in myelinating oligodendrocytes and CNS myelin sheaths. Anti-BP serum and anti-PLP serum were useful immunohistochemical staining reagents for the identification of myelinating oligodendrocytes and CNS myelin sheaths in tissue culture.     

Synaptogenesis and myelination in dissociated cerebral microcarrier cell cultures

A new approach for cultivating dissociated cerebral neurons is described. It is based on a rapid attachment of neurons to the DEAE-cellulose cylindrical MC and their subsequent interconnection to form cell-MC conglomerates. Intensive fiber growth followed by synaptogenesis and progressive myelin formation are indicative of optimal conditions of nutrition and oxygenation.     

Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures

Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury.     

Matrix metalloproteinase-2 is involved in myelination of dorsal root ganglia neurons

Matrix metalloproteinases (MMPs) comprise a large family of endopeptidases that are capable of degrading all extracellular matrix components. There is increasing evidence that MMPs are not only involved in tissue destruction but may also exert beneficial effects during axonal regeneration and nerve remyelination. Here, we provide evidence that MMP-2 (gelatinase A) is associated with the physiological process of myelination in the peripheral nervous system (PNS). In a myelinating co-culture model of Schwann cells and dorsal root ganglia neurons, MMP-2 expression correlated with the degree of myelination as determined by immunocytochemistry, zymography, and immunosorbent assay. Modulation of MMP-2 activity by chemical inhibitors led to incomplete and aberrant myelin formation. In vivo MMP-2 expression was detected in the cerebrospinal fluid (CSF) of patients with Guillain-Barré syndrome as well as in CSF and sural nerve biopsies of patients with chronic inflammatory demyelinating polyneuropathy. Our findings suggest an important, previously unrecognized role for MMP-2 during myelination in the PNS. Endogenous or exogenous modulation of MMP-2 activity may be a relevant target to enhance regeneration in demyelinating diseases of the PNS.     

Galanin increases membrane excitability and enhances Ca(2+) currents in adult, acutely dissociated dorsal root ganglion neurons

We examined the effect of galanin (10(-15) - 10(-7) M) on dispersed, mainly small-sized dorsal root ganglion (DRG) neurons in adult rats using whole-cell patch-clamp. Galanin and AR-M1896, a selective galanin type 2 receptor (GalR2) agonist, both significantly increased the number of action potentials in response to current pulses in 77% of the neurons, indicating an increase in excitability. Galanin also caused a rise in input resistance, decreased the holding current for -60 mV and depolarized the resting potential. In addition, Ca(2+) currents elicited by voltage steps were significantly increased by both galanin and AR-M1896 in nearly 70% of the cells. This enhancement was observed in 30% of the neurons in the presence of nimodipine or omega-conotoxin, but in each case approximately 60% less than without blocking either N- or L-type Ca(2+) channels, indicating modulation of both types of Ca(2+) channels. The percentage of small- and medium-sized neurons expressing GalR2 mRNA in DRGs in situ was similar to that showing increased excitability and Ca(2+) current after galanin application, i.e. approximately 70-80% of the neurons. The findings suggest that GalR2 has a role in controlling both the excitability, probably by inhibition of GIRK or leak K(+) channels, and Ca(2+) entry in a large population of presumably nociceptive neurons. The combination of the two effects, which possibly arise from separate biochemical pathways, would increase excitability and enhance intracellular Ca(2+) signalling which would enhance sensory transmission. These mechanisms involving GalR2 receptors may underlie the pronociceptive effects of galanin described in the literature.     

Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons

Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. ATP can elicit the sensation of pain. This study observed the effects of TMP on ATP-activated current (IATP) in rat DRG neurons. TMP (0.1-1 mM) concentration-dependently inhibited ATP (100 microM)-activated current in rat DRG neurons. The inhibitory time of ATP (100 microM)-activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s. The dose-response curves for IATP in the absence and presence of 1 mM TMP showed that TMP (1 mM) shifted the concentration-response curve of IATP downward markedly and the two EC50 values were very close (75 vs. 82 microM), while the threshold value remained unchanged. Therefore, the inhibitory effect of TMP on IATP may be noncompetitive. TMP did not alter the reversal potential (0 mV) of ATP-activated current, indicating that the site of TMP action is on or near the exterior surface of channel protein and not within the channel pore. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (PKC inhibitor) contained in pipette solution on IATP. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation via acting on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors.     

Ontogeny of calretinin in chick dorsal root ganglion neurons.

Calretinin immunostaining was performed on chick lumbosacral dorsal root ganglia during embryonic development. Calretinin-immunopositive neurons were first observed at around the 9th day of incubation. Quantitative evaluation revealed a close correlation between the number of immunopositive cells and the duration of incubation. Morphometric measurements disclosed that calretinin-immunoreactive cells belong in the large or intermediate categories of dorsal root ganglion neurons. It was concluded that the appearance of calretinin immunopositivity in spinal ganglion cells during development may be associated with both the morphological and functional maturation of this particular population of primary sensory neurons.     

MAG-deficient schwann cells myelinate dorsal root ganglion neurons in culture

The myelin-associated glycoprotein (MAG) has been postulated to play a crucial role during myelin formation. Evidence supporting this hypothesis was provided by infecting rat Schwann cells with a retrovirus expressing MAG antisense RNA; these Schwann cells showed reduced levels of MAG expression and failed to myelinate DRG neurons in vitro. However, when MAG expression was disrupted by generating MAG-deficient mice, normal myelin sheaths were formed in peripheral nerves in vivo. In the present study we investigated whether myelination is compromised in MAG-deficient Schwann cells in vitro, i.e., under similar conditions where Schwann cells expressing MAG antisense RNA failed to myelinate. We show that MAG-deficient Schwann cells do myelinate DRG neurons in vitro and express the myelin-specific glycolipid galactocerebroside (Gal-C) and the myelin proteins P0 and MBP. Furthermore, myelin sheaths appear morphologically normal with both compacted and uncompacted aspects when investigated by electron microscopy. Quantitative analysis revealed that the number of myelin sheaths was similar in cultures from MAG-deficient and wild-type mice. These findings support the view that MAG is not essential for myelin formation in the PNS. GLIA 22:213–220, 1998. © 1998 Wiley-Liss, Inc.