Adult mouse dorsal root ganglia neurons in cell culture

A method has been developed for the long-term culture of dissociated adult mouse dorsal root ganglia (DRG). Of critical importance to the success of this technique was a three-hour incubation in collagenase which softened the DRG and permitted gentle dissociation. The morphological and electrophysiological features of the dissociated adult DRG were similar to those observed in previous studies of immature (i.e., embryonic and newborn) DRG in culture and also to those of adult DRG in situ. With regard to electrophysiological work, the adult DRG neurons are superior to embryonic and newborn neurons because of their larger size and greatly increased survival in culture (no degeneration for first six days, and thereafter a relatively slow decrease). The adult neurons regenerated nerve fibers to an extent comparable to that of immature neurons. Therefore, the adult DRG cultures might be useful to study factors influencing regeneration in the adult mammalian nervous system. The adult cultures might also be useful to investigate factors influencing the aging process.     

Radiation-induced apoptosis in dorsal root ganglion neurons

Ionizing radiation (IR) results in apoptosis in a number of actively proliferating or immature cell types. The effect of IR on rat dorsal root ganglion (DRG) neurons was examined in dissociated cell cultures. After exposure to IR, embryonic DRG neurons, established in cell culture for six days, underwent cell death in a manner that was dose-dependent, requiring a minimum of 8 to 16 Gy. Twenty-five per cent cell loss occurred in embryonic day 15 (E-15) neurons, grown in cell culture for 6 days (“immature”), and then treated with 24 Gy IR. In contrast, only 2% cell loss occurred in E-15 neurons maintained in culture for 21 days ("mature") and then treated with 24 Gy IR. Staining with a fluorescent DNA-binding dye demonstrated clumping of the nuclear chromatin typical of apoptosis. Initiation of the apoptosis occurred within 24 h after exposure to IR. Apoptosis was prevented by inhibition of protein synthesis with cycloheximide. Apoptosis induced by IR occurred more frequently in immature than in mature neurons. Immature DRG neurons have a lower concentration of intracellular calcium ([Ca2+]i) than mature neurons. Elevation of [Ca2+]i by exposure to a high extracellular potassium ion concentration (35 μM) depolarizes the cell membrane with a resultant influx of calcium ions. The activation of programmed cell death after nerve growth factor (NGF) withdrawal is inversely correlated with [Ca2+]i in immature DRG neurons. When treated with high extracellular potassium, these immature neurons were resistant to IR exposure in a manner similar to that observed in mature neurons. These data suggest that [Ca2+]i modulates the apoptotic response of neurons after exposure to IR in a similar manner to that proposed by the “Ca2+ setpoint hypothesis” for control of NGF withdrawal-induced apoptosis.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

Long-term cultures of neurons from adult frog brain express GABA and glutamate-activated channels

Cells from adult Xenopus laevis brainstem and spinal cord were dissociated with mild enzymatic treatment and grown in long-term cell culture. These cells had specific attachment/substrate and medium/serum requirements. Cells with bipolar and multipolar morphology were positively identified as neurons using immunohistochemistry with monoclonal antibodies to rat and bovine neurofilament proteins which we show here cross-react with similar amphibian proteins. Patch clamp recordings demonstrated that these neurons have populations of ionic channels which are activated by L-glutamate or gamma-aminobutyric acid (GABA). The characteristics of these channels were similar to those previously described for GABA- and glutamate-activated channels in embryonic mammalian neurons isolated in culture. Cell cultures of neurons isolated from adult Xenopus laevis brain may be a useful and simple preparation with which to examine the modulation of neuronal properties by various agents over longer time intervals then has been previously possible.     

Structure and expression of differentiation antigens on functional subclasses of primary sensory neurons

Subpopulations of dorsal root ganglion neurons can be distinguished on the basis of their peripheral receptive properties, spinal terminal arbors and neuropeptide content. We have used monoclonal antibodies (MAbs) to define antigenic determinants on functional populations of DRG neurons projecting to the superficial dorsal horn of the spinal cord. Three MAbs recognize defined carbohydrate epitopes associated with lacto- and globo-series glycolipids that constitute the stage-specific embryonic antigens (SSEAs) 1, 3 and 4. SSEA-3 and SSEA-4 are present in the cytoplasm of about 10% of DRG neurons in adult rat. These neurons are distinct from those that contain substance P, somatostatin or the fluoride-resistant acid phosphatase enzyme, FRAP. SSEA-1 is present in a small percentage of DRG neurons. SSEAs are present on the surface of DRG neurons maintained in dissociated cell culture: 6% are SSEA-1+, 7% are SSEA-3+ and 10-15% are SSEA-4+. MAbs LD2, KH10, TC6 and TD10 identify epitopes expressed coincidently in 25% of small DRG neurons that project to lamina II of the dorsal horn. All somatostatin- but less than 1% of substance P-immunoreactive DRG neurons express these antigens. MAb LA4 labels a distinct population of small DRG neurons that also projects to lamina II. There is extensive overlap between LA4+ neurons and those that contain FRAP. Antigens recognized by these MAbs are expressed on the surface of 10-20% of DRG neurons in culture. Preliminary biochemical studies suggest that these antigens may be glycolipids. Molecules bearing carbohydrate differentiation antigens may be involved in the development and specification of sensory connections in the dorsal horn of the spinal cord.     

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.     

No evidence for effects of mild microwave irradiation on electrophysiological and morphological properties of cultured embryonic rat dorsal root ganglion cells

Effects of mild microwave treatment (1 hr, 37 degrees C) on the in vitro development of rat mechanically dissociated dorsal root ganglion (DRG) neurons were investigated to establish whether microwave irradiation effects exist on nervous tissue other than heat induced tissue fixation. Phase contrast microscopy and immunocytochemical neurofilament stainings did not reveal significant differences between irradiated (2 hr after isolation) and control cultures, maintained up till 21 days. The electrophysiological properties of microwave exposed and non-exposed DRG neurons were compared using the whole-cell patch-clamp technique. Control neurons, in culture for 0-12 days, were excitable. In cultured cells (1-12 days), microwaved 2 hr after isolation, the action potentials were similar to or slightly different from those of the control cells. No acute microwave effects were found on neurons irradiated after 1 day of culture. These results suggest that mild microwave irradiation has neither significant acute nor strong long-term effects on DRG culture development and DRG neuron membrane properties, consistent with the notion that microwave effects essentially are temperature effects.     

Evidence that nitric oxide-induced synthesis of cGMP occurs in a paracrine but not an autocrine fashion and that the site of its release can be regulated: studies in dorsal root ganglia in vivo and in vitro.

As nitric oxide is a gas, it cannot be stored and has to be synthesized as required. This suggests that it could be released wherever nitric oxide synthase (NOS) is activated and due to its unstable state will react with appropriate targets at this site of production. In both dissociated dorsal root ganglion (DRG) cultures and in acutely isolated, but intact, DRG, treatment with capsaicin or bradykinin caused cGMP synthesis, which could be blocked by NOS inhibitors. The cGMP was synthesized in cells different from those expressing the neuronal isoform of NOS (nNOS). In dissociated cultures many of the cells stimulated to produce cGMP were neurons, whereas in isolated ganglia they were always satellite glia cells. Surprisingly, the satellite glia cells surrounding the nNOS-containing neurons did not contain cGMP. Following nerve section in adult rats, many axotomized ganglion neurons expressed nNOS. Again in these axotomized ganglia, most cGMP was expressed in the satellite glia surrounding nNOS-negative neurons. However, an nNOS-selective inhibitor reduced the cGMP present in these axotomized ganglia, suggesting that the cGMP synthesized is stimulated by NO (nitrogen monoxide) produced by nNOS. In both dissociated cultures and axotomized ganglia, nNOS-containing processes were observed close to cGMP-positive cells. These observations lead to the suggestion that NO acts in a paracrine fashion when stimulating the synthesis of cGMP and may not be synthesized at all sites containing nNOS.     

Influence of factors released from sciatic nerve on adult dorsal root ganglion neurons

Previous experiments have shown that medium conditioned (CM) by denervated peripheral nerve contains a process outgrowth promoting factor (s) for cultured adult frog dorsal root ganglion (DRG) neurons. The present experiments further characterize the influences of these factors on DRG neurons. The growth factors increases average process length by threefold, restricts the number of processes extended from four to two while simultaneously altering the morphology of those processes. Neurons with preexisting processes respond to the factors by significantly increasing the length of 35% of these processes. Only the newly elongated portions of preexisting processes have morphology typical of factor-induced processes, while the previously extended portions retain their original morphology. The number of processes of these neurons remains unchanged. Although composed of two population according to size, neurons in both populations are similarly influenced, suggesting that the factors influence neurons of all sensory modalities. To look at other possible influences of the nerve-released factors, a novel simple culture system has been developed in which concentration gradients of these factors can be established and maintained. The front of the outgrowth-promoting influence in these cultures could be followed over time (up to 9 days) as it affected the process length and morphology of neurons at increasing distances (up to 8 mm) from the source of the factors. The trophic factors may play important roles during regeneration in vivo by influencing the cytoskeletal organization in the cell body and growth cones to bring about a stabilization and consolidation of growth cone membrane of only a limited number of processes resulting in increasing the rate of process elongation. The factors may also serve to direct process outgrowth, which can be examined using the new culture system. 1994 John Wiley & Sons, Inc.     

Glutamine Synthetase-Containing Cells of the Dorsal Root Ganglion at Different Stages of Rat Ontogeny

In the present study, formation, location, and morphological features of glutamine synthetaseimmunopositive cells of the dorsal root ganglion (DRG) at different stages of prenatal and postnatal development of the rat was examined. It was demonstrated that small differentiating satellite cells containing glutamine synthetase were observed in the DRG close to sensory neurons on embryonic day 18. On embryonic day 19, the forming immunopositive glial cells were located around developing neurons of the DRG in accordance with topography, which is observed in newborn and adult animals. The averaged number of satellite cells per sensory neuron in mature and aging rats was calculated and it was found that this index did not change during aging.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.     

CNS glial cells support in vitro survival, division, and differentiation of dissociated olfactory neuronal progenitor cells.

Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with [3H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells.     

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against β-tubulin III and S-100 β, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 μm on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Carbonic anhydrase activity in primary sensory neurons

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.     

Segmental independence and age dependence of neurite outgrowth from embryonic chick sensory neurons

Targets in limb regions of the chick embryo are further removed from the dorsal root ganglia that innervate them compared with thoracic ganglion-to-target distances. It has been inferred that axons grow into the limb regions two to three times faster than into nonlimb regions. We tested whether the differences were due to intrinsic properties of the neurons located at different segmental levels. Dorsal root ganglia (DRG) were isolated from the forelimb, trunk, and hind limb regions of stage 25–30 embryos. Neurite outgrowth was measured in dissociated cell culture and in cultures of DRG explants. Although there was considerable variability in the amount of neurite outgrowth, there were no substantive differences in the amount or the rate of outgrowth comparing brachial, thoracic, or lumbosacral neurons. The amount of neurite outgrowth in dissociated cell cultures increased with the stage of development. Overall, our data suggest that DRG neurons express a basal amount of outgrowth, which is initially independent of target-derived neurotrophic influences; the magnitude of this intrinsic growth potential increases with stage of development; and the neurons of the DRG are not intrinsically specified to grow neurites at rates that are matched to the distance they are required to grow to make contact with their peripheral targets in vivo. We present a speculative model based on Poisson statistics, which attempts to account for the variability in the amount of neurite outgrowth from dissociated neurons. © 1995 John Wiley & Sons, Inc.     

Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium

Summary In this study, we have documented by morphological analysis, immunocytochemistry, and electrophysiology, the development of a culture system that promotes the growth and long-term survival of dissociated adult rat spinal cord neurons. This system comprises a patternable, nonbiological, cell growth-promoting organosilane substrate coated on a glass surface and an empirically derived novel serum-free medium, supplemented with specific growth factors (acidic fibroblast growth factor, heparin sulfate, neurotrophin-3, brain-derived neurotrophic factor, glial-derived neurotrophic factor, cardiotrophin-1, and vitronectin). Neurons were characterized by immunoreactivity for neurofilament 150, neuron-specific enolase, Islet-1 antibodies, electrophysiology, and the cultures were maintained for 4–6 wk. This culture system could be a useful tool for the study of adult mammalian spinal neurons in a functional in vitro system.