Embryonic Rat Dorsal Root Ganglion Research Articles

Schwann cell plasminogen activator is regulated by neurons

The synthesis and release of plasminogen activators (PAs) in co-cultures of embryonic rat dorsal root ganglion nerve cells (NCs) and Schwann cells (SCs) were examined by metabolic labeling, immunoprecipitation, immunodepletion, SDS-PAGE, zymography, and a two-step esterolytic assay. Metabolic labeling of SC cultures followed by immunoprecipitation of the conditioned medium (CM) demonstrated that cultured SCs synthesized and released tissue type PA (tPA). Failure of amiloride to inhibit PA activity in SCCM indicated that urokinase PA (uPA) was unlikely to contribute significantly to PA activity in SCCM. Experimental manipulation of the NCs and SCs suggested that NCs regulated SC derived PA. Total PA activity increased in SCCM 10-14-fold by 6 days after removal of NCs. Multiple molecular weight forms of PAs were detected by SDS-PAGE followed by zymography. A PA approximately 95 kDa was absent in co-cultures of SCs + NCs but prominent by 4 days postdenervation; PA approximately 50-70 kDa increased through 8 days postdenervation and PA approximately 25 kDa, present in SC + NC cultures, was absent 8 days after removal of NCs. Upon reintroduction of NCs to denervated cultures (SCs), the pattern of PAs detected in culture medium was transitional between innervated and denervated cultures. Immunodepletion experiments using conditioned medium from denervated SC cultures indicated that various molecular weight forms of PA detected in SCCM by zymography were immunologically related to tPA. These studies demonstrate that SCs synthesized and released tPA in a tissue culture model of peripheral nerve and that one mechanism for regulation of PA released by SCs was by association with NCs. This regulation occurred in cultures of both myelinating and nonmyelinating Schwann cells and thus was not dependent on the state of myelination.

Kinetics of production of a novel growth factor after peripheral nerve injury

In response to transection injury, the distal segment of sciatic nerve produces a soluble factor which stimulates neurite outgrowth from 15 day embryonic rat dorsal root ganglion (DRG) neurons, and PC12 cells. This activity enhances survival of large sensory neurons, promotes myelination and has been designated SN. The expression of SN, undetectable in the perineurium and proximal segments, occurs solely in the endoneurium distal to the site of permanent transection. When the distal portion is removed immediately after transection, homogenized and the supernatant tested, there is little neurite promoting activity in the normal nerve. For the first 10 days after transection the major soluble factor present in the distal segment is NGF. The amount of neurite promoting activity increases after 10 days and appears to plateau at 30–35 days while the proportion that is inhibited by anti-NGF decreases. In a competitive receptor binding assay, SN does not compete with 125I-NGF for receptors on either DRG or PC12 cells. Separation using polyacrylamide-agarose followed by HPLC demonstrates that SN migrates with polypeptides of molecular weights 17.2 and 19.1 kDa.

Adrenal Medullary Transplantation Into the Brain for Treatment of Parkinson's Disease: Clinical Outcome and Neurochemical Studies

Transplantation of adrenal medulla into the caudate nucleus as treatment for Parkinson's disease was performed in eight patients. Although our previous 6-month follow-up revealed early modest improvement, an extension of that follow-up to 1 year disclosed no additional gains in any patient. At the end of 1 year, only one patient could be categorized as moderately improved; three patients were mildly improved, and four patients were unimproved. The rationale for transplanting adrenal medulla was to reestablish a physiologic source of dopamine to the striatum. We measured cerebrospinal fluid (CSF) and plasma catecholamines and metabolites before and after transplantation. Conjugated dopamine (the predominant form of dopamine found in the CSF) and homovanillic acid (the major dopamine metabolite) were modestly and inconsistently increased in the CSF. Conjugated and free epinephrine and norepinephrine, as well as 3-methoxy-4-hydroxyphenylglycol concentrations were not increased in CSF after graft placement, an indication that the adrenal chromaffin cells were no longer producing high levels of these nondopamine catecholamines and metabolites. CSF cortisol concentrations were not increased after transplantation, compared with values from controls, consistent with low numbers of functioning adrenal cortical cells contaminating the graft (or poor survival). Posttransplantation CSF did not induce a neurotrophic effect in cell cultures of 15-day embryonic rat dorsal root ganglion or PC12 (rat pheochromocytoma) cell lines. Survival of samples of patients' adrenal medullary tissue for 2 weeks in tissue culture attested to the viability of the graft at the time of transplantation. The relative concentrations of dopamine to epinephrine or norepinephrine increased in these cultured adrenal medullary cells, presumably because of loss of the glucocorticoid influence on catecholamine synthesis. A wide variety of factors could have contributed to our failure to replicate the earlier impressive results of adrenal-to-brain transplantation reported by others. Continued transplantation studies in animal models of parkinsonism are necessary for better elucidation of these factors.

Expression of sensory neuron antigens by a dorsal root ganglion cell line, F-11

The F-11 cell line is a fusion product of embryonic rat dorsal root ganglion (DRG) cells with mouse neuroblastoma cell line N18TG-2 (Platika, D., Boulos, M.H., Baizer, L. and Fishman, M.C., Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3499–3503). F-11 cells were uniformly labelled using a monoclonal antibody (RT-97) to the 200 kDa subunit of neurofilament protein, which labels a subpopulation of adult rat DRG neurons. F-11 cells did not stain for antigenic markers of fibroblasts or Schwann/satellite cells which are also present in DRG. Monoclonal antibodies that recognize cell surface carbohydrates have been shown to label subpopulations of DRG neurons. The stage-specific embryonic antigens SSEA-3 and SSEA-4, and the antigen recognized by B23D8, were expressed by some F-11 cells but not by the neuroblastoma parent of the hybrid cells. SSEA-3 was expressed by about 20% of the F-11 cells, whereas 40–60% expressed SSEA-4 or the antigen recognized by B23D8. The stability of F-11 cell subpopulations for sensory antigen expression was demonstrated by isolating single cells and growing the progeny as clonal lines. In some subclones, nearly 100% of the cells stably expressed SSEA-4 and/or B23D8, or failed to stain with anti-SSEA-4, anti-SSEA-3, or B23D8 over 12 passages. Other subclones were unstable for the expression of these antigens. This study demonstrates the derivation of the F-11 cell line from sensory neurons but also indicates that multiple phenotypes of varying stability are present in this line. This information is important for the use of this line as a model for DRG neurons.

Effects of ethanol on rat schwann cell proliferation and myelination in culture

It is possible to treat dissociated embryonic rat dorsal root ganglia in culture to inhibit proliferation of all nonneuronal cells except Schwann cells. Neurons have been shown to produce a mitogenic stimulus for Schwann cells under these conditions. Additionally, myelin-competent neurons induce Schwann cells to elaborate myelin sheaths. Groups of sibling cultures were exposed to various nonlethal concentrations of ethanol (0, 43, 86, or 172 mM) for 4 wk. Cultures were assessed weekly by light microscopy in a blind fashion for evidence of Schwann cell proliferation and myelin formation. Ethanol adversely affected both Schwann cell proliferation and myelin formation in culture. No obvious differences in neuronal morphology were observed among the various groups of cultures by light or electron microscopy. These observations suggest that ethanol might interfere with Schwann cell proliferation and myelin formation in culture by one or both of the following means: a) inhibit neuronal production of signals for Schwann cell proliferation and myelination or b) impede Schwann cell responses to neuronal signals. Investigation of these possibilities in culture may provide insight into neuropathologic mechanisms operative in the fetal alcohol syndrome or alcohol-associated peripheral neuropathy in humans.

Biological activity of a new neuronal growth factor from injured peripheral nerve

In response to transection injury, the distal nerve segment produces a soluble neurite promoting factor (SN). In this study, the ability to support neuronal survival and differentiation have been studied. Embryonic rat dorsal root ganglion (DRG) neurons were plated out on collagen substrates and incubated in medium containing either SN or nerve growth factor (NGF). The number of surviving neurons was counted after I, 2, 4, 7, and 15 days in vitro. After fixation and staining, the diameter of the surviving neurons was measured. During the period of observation, 60.8 ± 5.8% of plated neurons survived in the presence of NGF and 90.5 ± 12.9% survived with SN ( P < 0.05). The mean of median neuronal cell diameter was 28 ± 2.7 μm with NGF and 34.2 ± 3.7 μm with SN, ( P < 0.01). This increased diameter was due to enhanced survival of 30–50 μm diameter neurons. In parallel experiments, the degree of myelination of DRG neurons by Schwann cells was assessed morphometrically. In the presence of SN there was an 86% increase in myelination compared with NGF which indicates that not only is the survival of neurons increased but they are able to become fully differentiated in the presence of SN.

Residual ethylene oxide in hollow fiber hemodialysis units is neurotoxic in vitro.

Ethylene oxide gas is used to sterilize plastic medical equipment including capillary flow dialysis membranes. To test whether ethylene oxide retained in the dialyzers might be neurotoxic, tissue culture medium was incubated in the blood compartment of dialyzers. Embryonic rat dorsal root ganglion neurons were then incubated in this medium for up to 5 days. During the first 24 hours axonal growth was normal. During the next 24-hour period varicosities appeared on axons, and after 4 days neuron cell bodies died. The pattern of degeneration was identical to that observed when cultures were exposed to an atmosphere containing 1 ppm ethylene oxide gas. Culture medium introduced into dialyzers after routine prerinsing still caused degeneration, which was not completely abolished even by a 10-liter rinse. When medium was exposed to identical dialyzers sterilized by gamma irradiation, no changes were seen in culture. Identical morphological changes were produced by using dialysis patient serum in the culture medium in place of the usual calf bovine serum. Such changes were never seen with control human serum. Since ethylene oxide is toxic to the human peripheral nervous system, it is proposed that ethylene oxide in dialyzers may contribute to the progressive neuropathy observed in patients on long-term hemodialysis.

Herpes simplex virus infection of the rat sensory neuron Effects of interferon on cultured cells

Embryonic rat dorsal root ganglion neurons were cultured in a two-chamber system allowing infection of neuritic extensions without exposure of neuronal cell bodies or vice versa. Herpes simplex virus type 1 was used to infect interferon-alpha and -beta treated or untreated neurons and the production of virus and interferon was assayed. Treatment of nerve cell bodies with interferon inhibited virus replication in a dose-dependent manner, whether virus was inoculated directly onto the nerve bodies or peripherally on the neuritic extensions. In contrast no antiviral effect was noted when neurities were treated with interferon suggesting possible lack of interferon receptors on neurites. On infection with herpes simplex virus the rat sensory neuron cultures did not produce interferon in amounts above the detection limit (0.5 units per ml) of the interferon assay used.

Colchicine effect on B-50/GAP43 phosphoprotein localization in rat dorsal root ganglion explants

In rat embryonic dorsal root ganglion explants stimulated by nerve growth factor, the neuron-specific phosphoprotein B-50 (GAP43) is primarily localized in the distal portion of outgrowing neurites. Addition of colchicine leads to a decrease in total amount of B-50 and a marked redistribution in the neurons. The data underscore the role of axonal transport in the concentration of B-50 in growth cones of growing neurites.

Astroglia-induced detachment of central neurons but astroglia-dependent growth of peripheral neurons in rat embryonic spinal cord primary cultures

In mixed primary cultures, intrinnsic neurons from embryonic mammalian brains degenerate secondary to their detachment from the substratum and this is caused by the under-growing co-cultured astroglia. In the present study we sought to find out whether or not perripheral neurons, sensory and motor neurons which reside and/or only project outside the CNS respectively, interact with astroglia similarly as their central counterparts do. Mixed primary cultures prepared from dissociated embryonic rat spinal cord and dorsal root ganglia were examined by phase and immunofluorescence microscopy after labeling with antibodies to neurofilaments (neuronal markers) and to glial fibrillary acidic protein and vimentin (astroglia markers). Acetylcholinesterase staining served as a marker for motor neurons. In this system astroglia grew exclusively under intrinsic neurons of the spinal cord and with time (about 8 days) all these neurons detached and disappeared. In contrast, astroglia were intimately associated with perikarya of peripheral neurons. sometimes growing over them. Furthermore, the neuritic processes of these neurons were attached to the undergrowing astroglia. Central neurons could be rescued by treatment of cultures with the antimitotic drug cytosine arabinoside which led to the elimination of astroglia. However, this treatment resulted in death of all peripheral neurons. We conclude: (1) survival of intrinsic CNS neurons in culture is independent of astroglia; (2) astroglia are responsible for the detachment of these neurons from their growth substratum; (3) survival of peripheral sensory and motoneurons is dependent on co-cultured astroglia and (4) the differences in neuron-astroglia interactions between central and peripheral neurons are membrane-associated and probably independent of soluble factors.

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.

Sulfated proteoglycans produced by rat dorsal root ganglion cells.

Primary organotypic cultures of embryonic rat dorsal root ganglia, which contain sensory neurons, Schwann cells, and fibroblasts, produce an extensive extracellular matrix. These cultures actively incorporated 35SO4 into glycosaminoglycans, of which 30% were heparan sulfate, 65% chondroitin sulfate, and 5% dermatan sulfate. Sulfate-labeled proteoglycans made by these cells were extracted with 4 M guanidine-hydrochloride and purified by CsCl density gradient centrifugation, gel filtration chromatography, and DEAE-cellulose chromatography. Eight individual species were resolved, of which five were subjected to further analysis. One low-density (less than 1.35 g/ml) proteoglycan, with a Kav on Sepharose Cl-4B = 0.34 contained heparan sulfate chains of Mr = 20,000. The other four proteoglycans, with Kav on Sepharose Cl-4B of 0.04, 0.35 (two), and 0.44, contained predominantly chondroitin sulfate chains with Mr ranging from 30,000 to 40,000. To determine possible functions of these proteoglycans, cultures were grown in medium containing 1 mM 4-methyl-umbelliferyl-beta-D-xyloside. The drug inhibited 35SO4 proteoglycan accumulation in the cell layer by approximately 90%. Drug-treated cultures exhibited several developmental abnormalities, including decreased migration of fibroblast-like cells, abnormal morphology of these cells, and decreased myelination of axons by Schwann cells.

Amino acid receptor-mediated transmission at primary afferent synapses in rat spinal cord.

Intracellular recording techniques have been used to provide information on the identity of excitatory transmitters released at synapses formed between dorsal root ganglion (DRG) and spinal cord neurones in two in vitro preparations. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurones and synaptic potentials recorded intracellularly from dorsal horn neurones after DRG explant stimulation. More than 80% of dorsal horn neurones received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations (5 mmol l-1 Ca2+, 3 mmol l-1 Mg2+) the acidic amino acid receptor agonists, L-glutamate, kainate (KA) and quisqualate (QUIS) excited all dorsal horn neurones which received a monosynaptic DRG neurone input, whereas L-aspartate and N-methyl-D-aspartate (NMDA) had little or no action. 2-Amino-5-phosphonovalerate (APV), a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and L-glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurones. Intracellular recordings from motoneurones in new-born rat spinal cord were used to study the sensitivity of the Ia excitatory postsynaptic potential (EPSP) to antagonists of excitatory amino acids. Superfusion of the spinal cord with APV did not inhibit the Ia EPSP but did suppress later, polysynaptic components of the afferent-evoked response. Kynurenate was a potent and selective inhibitor of the Ia EPSP, acting via a postsynaptic mechanism. These findings indicate that L-glutamate, or a glutamate-like compound, but not L-aspartate, is likely to be the predominant excitatory transmitter that mediates fast excitatory postsynaptic potentials at primary afferent synapses with both dorsal horn neurones and motoneurones.

Synaptic transmission between dorsal root ganglion and dorsal horn neurons in culture: antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate

Intracellular recording techniques have been used to provide information on the identity of excitatory sensory transmitters released at synapses formed between dorsal root ganglion (DRG) and dorsal horn neurons maintained in cell culture. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurons and synaptic potentials were recorded intracellularly from dorsal horn neurons after DRG explant stimulation. More than 80% of dorsal horn neurons within 1 mm of DRG explants received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations, the acidic amino acid receptor agonists, L-glutamate, kainate, and quisqualate excited all dorsal horn neurons which received a monosynaptic DRG neuron input, whereas aspartate and N-methyl-D-aspartate (NMDA) had little or no action. Several compounds reported to antagonize the actions of acidic amino acids were tested for their ability to block DRG-evoked synaptic potentials and glutamate-evoked responses in dorsal horn neurons. 2-Amino-5-phosphonovalerate, a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurons. These findings indicate that glutamate, or a glutamate-like compound, but not aspartate, is the excitatory transmitter that mediates fast excitatory postsynaptic potentials at the DRG-dorsal horn synapses examined in this study.

Neuronal traits of clonal cell lines derived by fusion of dorsal root ganglia neurons with neuroblastoma cells.

In an attempt to immortalize the gene products of single neurons, somatic cell hybrids were produced by fusion of embryonic rat dorsal root ganglion (DRG) neurons with mouse neuroblastoma cells. Embryonic day 13 rat DRGs were fused with mouse neuroblastoma cells deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8). The hybrid cells were selected in medium with 100 microM hypoxanthine/1 microM aminopterin/12 microM thymidine to eliminate the neuroblastoma cells and with cis-hydroxyproline to retard fibroblast growth. Of the 17 lines derived, 4 manifested neuronal properties and were cloned. These lines retain both rat and mouse chromosomes and synthesize characteristic rat and mouse isoenzymes. Neuronal gangliosides, action potentials, and extensive neurite-like processes are exhibited by these hybrid cells, properties characteristic of DRG neurons but not of the neuroblastoma parent. Each line manifests a unique combination of action-potential properties and cell-surface markers, suggesting the selective expression of subsets of DRG neuronal genes. All of these neuronal properties are expressed constitutively, without the need for chemical induction or mitotic inhibition, and stably, without diminution after at least 5 months in culture. These lines may prove useful in the identification and isolation of gene products that characterize individual or small subsets of DRG neurons.

Immunohistochemical localization of troponin-C in cultured neurons.

Our previous immunofluorescence studies on neurons have demonstrated the presence of myosin in regions of neurons which contained actin. To determine if a system similar to the troponin complex of striated muscle is present in neurons, antibody shown to be specific for the calcium-binding component of troponin (troponin-C) was applied to cultures of embryonic chick and rat dorsal root ganglia. Neurites treated with anti-troponin-C exhibited a bright fluorescence. Accompanying non-neuronal cells were less reactive than the neuronal elements. Immunodiffusion and immunofluorescence showed that the anti-troponin-C did not react with calmodulin, whereas homogenates of the ganglia elicited a positive immunochemical reaction with the anti-troponin-C in Ouchterlony tests. Our results suggest that some intra-axonal movements may be generated by the interaction of actin and myosin and controlled in part by a calcium-troponin-C-dependent mechanism.

Nerve growth factor stimulates development of substance P in the embryonic spinal cord

Development of the putative neurotransmitter, substance P (SP), in the embryonic rat dorsal root ganglion (DRG) and spinal cord was defined in vivo. SP was not detectable by radioimmunoassay before day 17 of gestation (E17). On E17, cervical sensory ganglia contained 4 pg SP/ganglion, rising to 49 pg/ganglion at birth. The dorsal cervical spinal cord contained 0.75 ng SP/mg protein on E17, rising to 6 ng SP/mg protein on postnatal day 3. The ventral spinal cord contained approximately 20% of the SP content in the dorsal spinal cord at each gestational age. Intrauterine forelimb amputation partially prevented the normal developmental increase of SP in sensory ganglia destined to innervate that limb, suggesting that target structures regulate the development of peptidergic neurons. Conversely, treatment with nerve growth factor (NGF) stimulated development of SP in the DRG. Moreover, NGF treatment increased SP in the dorsal spinal cord, suggesting that NGF can modulate development within the CNS, as well as peripheral structures. It is likely that the CNS effect reflects NGF action on peripheral ganglia, but a direct effect on the spinal cord has not been excluded. However, treatment with antiserum to NGF failed to significantly inhibit development of ganglion SP. The system of SP-containing neurons in the DRG may provide a convenient model for defining events regulating peptidergic maturation.

Ultrastructural Effects of Herpes Simplex Virus Type 2 Infection of Rat Dorsal Root Ganglia in Culture

The ultrastructural consequences of herpes simplex virus type 2 (strain R-2) infection of organotypic cultures of embryonic rat dorsal root ganglia were studied. The intial consequences (dilation of endoplasmic reticulum and/or Golgi apparatus and distortion of mitochondrial structure) occurred in the cytoplasm. These effects were followed by several nuclear changes including loss of nucleoplasm, and margination of chromatin. Extensive nuclear membrane proliferation was accompanied by the viral maturation process. Two previously unreported observations were made. First, productive virus replications occurred in glial cells, as well as in neurons. Mature, enveloped virus was produced by nuclear budding and envelopment in the cytoplasm in both cell types. Second, neurons were observed to participate in polykaryocyte formation with other neurons and with glial cells. These polykaryocytes were usually composed of only three or four cells. Neuronal-glial polykaryocytes were more prevalent than neuronal-neuronal polykaryocytes. In general, however, the ultrastructural changes in neurons and glial cells in culture were consistent with previously reported changes occurring in nervous tissue of experimental animals suffering from acute herpes simplex virus infections. Therefore, the utilization of this in vitro system to further study the pathogenesis of acute herpetic infections of sensory ganglia appears to be reasonable.