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.     

Restrictions of developmental capacities in the dorsal root ganglia during the course of development

By grafting ganglia from embryonic quails into the neural crest migration pathway of 2-day chick embryos, it was previously demonstrated that all type of ganglia possess more developmental potentialities than those normally expressed in the normal course of development. Namely autonomic neurones with catecholamine and adrenomedullary cells can be obtained from grafted spinal ganglia. The latter also yield sensory neurons to the host dorsal root ganglia (DRG) but only if they are taken from the donor before 8 days of incubation. In the present article we show that the capacity to differentiate sensory neurons in back-transplantation experiments can be correlated with the presence in the donor DRG of cycling neuronal precursors. Once all the neurons have been withdrawn from the cell cycle - an event which occurs first in the mediodorsal and then in the lateroventral area of the ganglion - the DRG cell population gives rise exclusively to autonomic ganglion cells in the host. It is concluded that in the conditions of the back-transplantation experiments, the postmitotic neurons contained in the donor ganglion do not survive. Therefore, the neurons and paraganglion cells which differentiate in the host arise from still undifferentiated precursor cells. This indicates that besides sensory neuron precursors the embryonic DRG cell population also contains precursor cells for the autonomic differentiation pathway.     

Viability and Isolation of Marine Bacteria by Dilution Culture: Theory, Procedures, and Initial Results

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10⁴ or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10⁴ cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.     

Threshold-free population analysis identifies larger DRG neurons to respond stronger to NGF stimulation

Sensory neurons in dorsal root ganglia (DRG) are highly heterogeneous in terms of cell size, protein expression, and signaling activity. To analyze their heterogeneity, threshold-based methods are commonly used, which often yield highly variable results due to the subjectivity of the individual investigator. In this work, we introduce a threshold-free analysis approach for sparse and highly heterogeneous datasets obtained from cultures of sensory neurons. This approach is based on population estimates and completely free of investigator-set parameters. With a quantitative automated microscope we measured the signaling state of single DRG neurons by immunofluorescently labeling phosphorylated, i.e., activated Erk1/2. The population density of sensory neurons with and without pain-sensitizing nerve growth factor (NGF) treatment was estimated using a kernel density estimator (KDE). By subtraction of both densities and integration of the positive part, a robust estimate for the size of the responsive subpopulations was obtained. To assure sufficiently large datasets, we determined the number of cells required for reliable estimates using a bootstrapping approach. The proposed methods were employed to analyze response kinetics and response amplitude of DRG neurons after NGF stimulation. We thereby determined the portion of NGF responsive cells on a true population basis. The analysis of the dose dependent NGF response unraveled a biphasic behavior, while the study of its time dependence showed a rapid response, which approached a steady state after less than five minutes. Analyzing two parameter correlations, we found that not only the number of responsive small-sized neurons exceeds the number of responsive large-sized neurons--which is commonly reported and could be explained by the excess of small-sized cells--but also the probability that small-sized cells respond to NGF is higher. In contrast, medium-sized and large-sized neurons showed a larger response amplitude in their mean Erk1/2 activity.     

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.     

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

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.     

瞬时外向钾电流的降低介导了慢性压迫背根神经节细胞兴奋性的升高

慢性压迫大鼠背根神经节（chronic compression of the dorsal root,ganglion,CCD）后,背根神经节细胞兴奋性升高,但引起神经元兴奋性改变的离子通道机制还需进一步探索。本实验采用胞内记录以及全细胞膜片钳记录方法,研究急性分离的大鼠背根神经节细胞兴奋性改变与瞬时外向钾电流（A-type potassium current,ⅠA）的关系。结果表明,CCD术后背根神经节细胞兴奋性升高,在急性分离的体外细胞中仍继续存在,表现为对辣椒素敏感的背根神经节细胞产生动作电位的最小电流刺激强度,即阈电流（current threshold）及阈电位（voltage threshold）降低;给予正常对照组神经元（未压迫损伤）瞬时外向钾通道阻断剂4-氨基吡啶,出现了类似CCD术后兴奋性升高的改变。进一步用两步电压钳方法分离ⅠA,研究CCD术后神经元ⅠA的变化,结果表明,CCD组神经元的ⅠA比对照组神经元ⅠA降低,并且与其阈电位的改变一致。以上结果提示,背根神经节压迫受损后,神经节细胞ⅠA降低可能参与介导了神经节细胞兴奋性的升高。     

Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem Cells

The dissociation of adherent mesenchymal stem cell (MSC) monolayers with trypsin and enzyme-free dissociation buffer was compared. A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin. Subsequently, the dissociated cells were re-seeded on new cell culture dishes and were subjected to the MTT assay 24 h later. The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin. Frozen–thawed MSC displayed a similar trend, yielding consistently higher cell viability and reattachment rates when dissociated with trypsin compared to enzyme-free dissociation buffer. It was also demonstrated that exposure of trypsin-dissociated MSC to enzyme-free dissociation buffer for 1 h had no significant detrimental effect on cell viability.     

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 investigated factors influencing the aging process.     

Glutamate receptor channels in rat DRG neurons: activation by kainate and quisqualate and blockade of desensitization by Con A

Primary afferent C fibers in rat dorsal roots are depolarized by the excitatory amino acids kainate and domoate. Under whole-cell voltage clamp, kainate and domoate increase membrane conductance in a subpopulation of freshly dissociated DRG neurons. In contrast to kainate currents observed in CNS neurons, responses to kainate and domoate in DRG cells desensitize with prolonged agonist exposure. Half-maximal activation is achieved with much lower concentrations of kainate and domoate in sensory neurons than in CNS neurons from cerebral cortex. Rapid applications of glutamate, quisqualate, and AMPA evoke a transient current in DRG neurons and desensitize cells to subsequent applications of kainate or domoate. Brief incubation with the lectin concanavalin A eliminates desensitization to excitatory amino acids; after treatment with concanavalin A, all five agonists gate sustained currents of similar amplitude via the same receptor.     

An Efficient Method for Dorsal Root Ganglia Neurons Purification with a One-Time Anti-Mitotic Reagent Treatment

Background

The dorsal root ganglia (DRG) neuron is an invaluable tool in axon growth, growth factor regulation, myelin formation and myelin-relevant researches. The purification of DRG neurons is a key step in these studies. Traditionally, purified DRG neurons were obtained in two weeks after exposure to several rounds of anti-mitotic reagent.

Methods and Results

In this report, a novel, simple and efficient method for DRG purification is presented. DRG cultures were treated once with a high-dose anti-mitotic reagent cocktail for 72 hours. Using this new method, DRG neurons were obtained with 99% purification within 1 week. We confirmed that the neurite growth and the viability of the purified DRG neurons have no difference from the DRG neurons purified by traditional method. Furthermore, P0 and MBP expression was observed in myelin by immunocytochemistry in the DRG/SC co-culture system. The formation of mature node of Ranvier in DRG-Schwann cell co-culture system was observed using anti-Nav 1.6 and anti-caspr antibody.

Conclusion and Significance

The results indicate that this high dose single treatment did not compromise the capacity of DRG neurons for myelin formation in the DRG/SC co-culture system. In conclusion, a convenient approach for purifying DRG neurons was developed which is time-saving and high-efficiency.     

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.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

The effect of acute dissociation on the electrophysiological properties of rat dorsal root ganglion neurons

The acutely dissociated neurons from the dorsal root ganglia (DRGs) are extensively used. The effects of acute dissociation on the properties of these neurons are, however, not clear. In this study, the action potentials (APs) were recorded from both acutely dissociated and in vivo identified DRG neurons with patch clamp and sharp electrode recording techniques, respectively. We found that acute dissociation slowed both the depolarizing and repolarizing rate of APs, and elongated the AP duration (APD). The lower recording temperature presented in the acutely dissociated neurons contributed to about 10% of these differences. The major contributor of these differences was possibly modulation of the mRNA expression especially those of the ion channels, as suggested by our observation that acute dissociation significantly reduced the mRNA abundance of Nav1.6–1.9. In conclusion, acute dissociation altered the electrophysiological properties of the DRG neurons; the disrupted gene-expression pattern may contribute to this effect.     

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.     

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.     

A strategy for an improved separation of mammalian spermatids

A collection procedure has been developed to improve the homogeneity of mammalian spermatid populations separated by elutriation. Trypsinizied ram testis cells were elutriated at 18C. Every cell population was eluted by progressive changes in the flow rate and/or rotor speed, instead of by abrupt changes, to reduce the contamination by cells from the next population. Pure populations were collected alternating with mixed populations corresponding to the overlap between two adjacent pure populations. Furthermore, each pure population was collected into two subfractions, the second of which, contamined by cells from the following population, was pooled with the following fraction. In less than 2 hr after castration, three populations of at least 1 × 10⁸ viable round or elongated or elongating spermatids were obtained with respective purities of 95%, 82%, and 99% of the nucleated cells. In addition, two mixed populations containing only two adjacent spermatid types (round plus elongating spermatids: 98%; elongated plus elongating spermatids: 98%) were obtained, as well as a population containing around 60% pachytene spermatocytes.