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.     

大鼠初级感觉神经元P2X3受体的表达及其与SP的关系

目的研究在大鼠初级感觉神经元细胞上P2X3受体的表达情况及其与P物质的关系。方法取SD大鼠背根神经节(DRG)和三叉神经节(TG)固定后切片;用抗P2X3受体抗体和抗SP抗体进行免疫组织化学反应,并通过两种不同的显色方法同时进行P2X3受体和SP的双标。结果P2X3免疫反应阳性细胞主要集中在小细胞和中等细胞(其中在TG,P2X3-ir阳性神经元约占整个细胞的24.8%;在DRG约31.7%的神经元是P2X3-ir阳性),并且在DRG和TG细胞上均存在有P2X3受体和SP共存(TG上的双标细胞占P2X3-ir阳性细胞总数的36.26%,DRG上占46.81%)。结论由于ATP门控阳离子通道受体P2X3本身就与伤害性感受的初级传入有关,而它与SP的共存可提示当组织中的ATP释放时可以通过P2X3受体作用于含SP的伤害性感觉神经末梢上,促使SP释放引起痛觉过敏。     

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal trasnport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine wheter the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se. © 1993 John Wiley & Sons, Inc.     

A monoclonal antibody (SN1) identifies a subpopulation of avian sensory neurons whose distribution is correlated with axial level

We have produced a monoclonal antibody, designated SN1, which binds to the surfaces of a subpopulation of avian sensory neurons, but not to other neurons of the peripheral or central nervous systems. The proportion of SN1(+) neurons in brachial and lumbosacral dorsal root ganglia (DRG), which innervate the wings and legs respectively, is low (30-40%), compared to the proportion (80-90%) in the lower thoracic DRG. SN1 immunoreactive fibers project to laminae I and II of the spinal cord dorsal horn, and are seen in the skin, but not the deeper tissues of older embryos. On the basis of the time of appearance, axial level-dependent distribution, and the central and peripheral projections of SN1(+) neurons, we suggest that they are cutaneous afferents that depend on interaction with peripheral targets to differentiate.     

Infection of human fetal dorsal root ganglion glial cells with human immunodeficiency virus type 1 involves an entry mechanism independent of the CD4 T4A epitope.

Human immunodeficiency virus type 1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome-associated neurological dysfunction, and it is believed that the presence of CD4 in the nervous system may be involved in the susceptibility of selected neural cell populations to HIV-1 infection. We previously demonstrated (B. Wigdahl, R. A. Guyton, and P. S. Sarin, Virology 159:440-445, 1987) that glial cells derived from human fetal dorsal root ganglion (DRG) are susceptible to HIV-1 infection and subsequently express at least a fraction of the virus genome. In contrast to HIV-1 infection of CD4+ lymphocytes, which can be blocked by treatment with monoclonal antibodies directed against the HIV-1-binding region of CD4 (T4A epitope), treatment of human fetal DRG glial cells with similar antibodies resulted in only a slight reduction in HIV-1-specific gag antigen expression. In addition, preincubation of the HIV-1 inoculum prior to infection with HIV-1-neutralizing antiserum did not reduce HIV-1 gag antigen expression in these cells. Furthermore, we were unable to detect the synthesis or accumulation of the CD4 molecule in neural cell populations derived from DRG. However, a protected CD4-specific RNA fragment was detected in RNA isolated from human fetal DRG and spinal cord tissue by an RNase protection assay with a CD4-specific antisense RNA probe. RNA blot hybridization analysis of total cellular RNA isolated from human fetal DRG and spinal cord demonstrated specific hybridization to an RNA species that comigrated with the mature 3.0-kilobase CD4 mRNA as well as two unique CD4 RNA species with relative molecular sizes of approximately 5.3 and 6.7 kilobases. Furthermore, all three CD4-related RNA species were polyadenylated when isolated from human fetal spinal cord tissue. These data suggest that HIV-1 infection of human fetal DRG glial cells may proceed via a mechanism of viral entry independent of the T4A epitope of CD4.     

Identification of alpha-galactose (alpha-fucose)-asialo-GM1 glycolipid expressed by subsets of rat dorsal root ganglion neurons

Distinct cell-surface glycoconjugates are expressed on specific subsets of dorsal root ganglion (DRG) neurons and DRG terminals projecting to the superficial dorsal horn of rat spinal cord (Dodd, J., and Jessell, T. M. (1985) J. Neurosci. 5, 3278-3294). Carbohydrate antigens detected by monoclonal antibodies (mAbs) TC6, KH10, and LD2 are restricted to about 20% of DRG neurons projecting to lamina IIB (dorsal), whereas antigens recognized by mAb LA4 are expressed by about 50% of DRG neurons projecting to lamina IIB (ventral). These mAbs were generated against rat pancreatic acinar cell line AR4-2J antigens. The glycolipid antigens in AR4-2J cells reacting with these mAbs have been structurally characterized by sequential hydrolysis with various exoglycosidases, immunochemical tests, linkage analysis of permethylated alditol acetates, capillary gas liquid chromatography-mass spectrometry, mass spectrometry of permethylated compounds, and by fast atom bombardment mass spectrometry of the native antigens. The structure of the major antigen (IA) in AR4-2J cells was determined to be: (formula; see text) The asialo derivative of IA and the novel disialo form of IA (Gal alpha 1----3(Fuc alpha 1----2)----GD1b) have been also identified. The DRG neurons contained only the neutral glycolipid, asialo form of IA. All these antigens reacted equivalently in the high performance thin layer chromatography-immuno overlay assay with the TC6, LD2, and LA4 mAbs. The molecular specificity of the three mAbs was determined by rection with a variety of possible antigens and appears to be the same. All three mAbs required terminal Gal alpha 1----3(Fuc alpha 1----2)Gal beta 1----3GalNAc (or 4GlcNAc) for full reactivity. Only partial reactivity was observed with compounds in which alpha-Fuc was removed. The observed restricted reactivity of mAbs TC6, LD2, and LA4 in subsets of DRG neurons and in ventral and dorsal areas of lamina IIB may be due to different topographical expression of the antigen in the neuronal membrane.     

Schwann cells promote neurite outgrowth of dorsal root ganglion neurons through secretion of nerve growth factor

The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.     

The interaction of human fetal neurons and epidermal cellsin vitro

Summary the interaction of autologous human fetal neurons with epidermal cells was studied by culturing fetal dorsal root ganglia (DRG) in the center of a dual chamber system with epidermal explants in the outer chamber. The two chambers were separated by two concentric stainless steel annular rings adherent to the substratum by silicon grease and agarose. Axons from the DRG penetrated the agarose barrier, growing into the exterior chamber by 10 din vitro (DIV) and extended past sparse peripheral fibroblasts to interact specifically with epidermal cells by 12 to 16 DIV. Scanning electron microscopy (SEM) showed single or multiple neuronal fascicles terminating on epidermal cells with spatular, veillike or bulbous axon termini. Transmission electron microscopy (TEM) showed fine axonal termini between epidermal cells, separated by an intercellular gap. The specificity of axonal targeting for epidermal cells rather than fibroblasts was also demonstrated by infecting the DRG with Herpes simplex virus type-1 (HSV-1). Specific anterograde transport of HSV-1 along axons to keratin-expressing epidermal cells was demonstrated by immunofluorescence and immunoperoxidase staining using monoclonal antibodies to viral glycoprotein D. This model allows the study of the mechanism of the specific interactions between neurons and epidermal cells analogous to those in fetal development and after cutaneous nerve regeneration.     

Antibodies against mouse nerve growth factor interfere in vivo with the development of avian sensory and sympathetic neurones

The monoclonal antibody 27/21 directed against mouse nerve growth factor (NGF) interferes in vivo with the survival of sensory dorsal root ganglion (DRG) neurones during the development of the quail embryo: the number of DRG neurones at embryonic day 11 (E11) was reduced by about 30% in embryos treated with the antibody between E3 and E11. Neurone numbers in the nodose ganglion were not affected. The effect of NGF antibodies on sympathetic neurones was assessed by determining the levels of the adrenergic marker enzyme tyrosine hydroxylase. Both total tyrosine hydroxylase activity and protein levels in sympathetic chains were reduced by about 30% in embryos treated with 27/21 antibody but not in embryos treated with a control antibody. The 27/21 antibody cross-reacts with chick NGF-like activity as shown in vitro by the ability of the antibody to partially block the survival activity of chick-embryo-fibroblast-conditioned medium for E9 chick DRG neurones.     

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.     

Mouse colon sensory neurons detect extracellular acidosis via TRPV1

Extracellular acidification contributes to pain by activating or modulating nociceptor activity. To evaluate acidic signaling from the colon, we characterized acid-elicited currents in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglion (DRG) neurons identified by content of a fluorescent dye (DiI) previously injected into the colon wall. In 13% of unidentified LS DRG neurons (not labeled with DiI) and 69% of LS colon neurons labeled with DiI, protons activated a sustained current that was significantly and reversibly attenuated by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine. In contrast, 63% of unidentified LS DRG neurons and 4% of LS colon neurons exhibited transient amiloride-sensitive acid-sensing ion channel (ASIC) currents. The peak current density of acid-elicited currents was significantly reduced in colon sensory neurons from TRPV1-null mice, supporting predominant expression of TRPV1 in LS colon sensory neurons, which was also confirmed immunohistochemically. Similar to LS colon DRG neurons, acid-elicited currents in TL colon DRG neurons were mediated predominantly by TRPV1. However, the pH producing half-activation of responses significantly differed between TL and LS colon DRG neurons. The properties of acid-elicited currents in colon DRG neurons suggest differential contributions of ASICs and TRPV1 to colon sensation and likely nociception. visceral pain; dorsal root ganglion neurons; acid-sensing ion channel; capsaicin receptor; acid-evoked currents; transient receptor potential vanilloid receptor 1     

The majority of dorsal spinal cord gastrin releasing peptide is synthesized locally whereas neuromedin B is highly expressed in pain- and itch-sensing somatosensory neurons

ABSTRACT: BACKGROUND: Itch is one of the major somatosensory modalities. Some recent findings have proposed that gastrin releasing peptide (Grp) is expressed in a subset of dorsal root ganglion (DRG) neurons and functions as a selective neurotransmitter for transferring itch information to spinal cord interneurons. However, expression data from public databases and earlier literatures indicate that Grp mRNA is only detected in dorsal spinal cord (dSC) whereas its family member neuromedin B (Nmb) is highly expressed in DRG neurons. These contradictory results argue that a thorough characterization of the expression of Grp and Nmb is warranted. FINDINGS: Grp mRNA is highly expressed in dSC but is barely detectable in DRGs of juvenile and adult mice. Anti-bombesin serum specifically recognizes Grp but not Nmb. Grp is present in a small number of small-diameter DRG neurons and in abundance in layers I and II of the spinal cord. The reduction of dSC Grp after dorsal root rhizotomy is significantly different from those of DRG derived markers but similar to that of a spinal cord neuronal marker. Double fluorescent in situ of Nmb and other molecular markers indicate that Nmb is highly and selectively expressed in nociceptive and itch-sensitive DRG neurons. CONCLUSION: The majority of dSC Grp is synthesized locally in dorsal spinal cord neurons. On the other hand, Nmb is highly expressed in pain- and itch-sensing DRG neurons. Our findings provide direct anatomic evidence that Grp could function locally in the dorsal spinal cord in addition to its roles in DRG neurons and that Nmb has potential roles in nociceptive and itch-sensitive neurons. These results will improve our understanding about roles of Grp and Nmb in mediating itch sensation.     

Morphological and functional properties of rat dorsal root ganglion cells cultured in a chemically defined medium

A culture procedure for dorsal root ganglion (DRG) cells is presented using a completely defined culture medium without antibiotics, in combination with mechanical dissociation procedures. This culture procedure allows all dorsal root ganglion cell types to be cocultured for periods of at least 106 days. Some of the dorsal root ganglion neurons, which could be identified by their neurofilaments and the presence of fluoride resistant acid phosphatase, regained their original T-cell appearance within two weeks. After one month in culture ganglion-like reaggregates appeared. Schwann cells, satellite cells and fibroblasts were identified using morphological criteria. All neurons tested maintained excitability during, at least, the first 35 days in culture, since in all cases action potentials could be evoked by current pulses. The method has proved to be useful in the study of morphological, cytochemical and electrophysiological aspects of dorsal root ganglion cell differentiation in vitro.     

Analysis of glia cell differentiation in the developing chick peripheral nervous system: sensory and sympathetic satellite cells express different cell surface antigens.

To identify and analyse precursor cells of neuronal and glial cell lineages during the early development of the chick peripheral nervous system, monoclonal antibodies were raised against a population of undifferentiated cells of E6 dorsal root ganglia (DRG). Non-neuronal cells of E6 DRG express surface antigens that are recognized by four monoclonal antibodies, G1, G2, GLI 1 and GLI 2. The proportion of non-neuronal cells in DRG that express the GLI 1 antigen is very high during ganglion formation (80% at E4) and decreases during later development (15% at E14). GLI 2 antigen is expressed only on a minority of the cells at E6 and increases with development. The G1 and G2 antigens are expressed on about 60-80% of the cells between E6 and E14. All cells that express the established glia marker O4 are also positive for the new antigens. In addition, it was demonstrated that GLI 1-positive cells from early DRG, which are devoid of O4 antigen, could be induced in vitro to express the O4 antigen. Thus, the antigen-positive cells are considered as glial cells or glial precursor cells. Surprisingly, the antigen expression by satellite cells of peripheral ganglia is dependent on the type of ganglion: antigens G1, G2 and GLI 1 were not detectable on glial cells of lumbosacral sympathetic ganglia and GLI 2 was expressed only by a small subpopulation. These results demonstrate an early immunological difference between satellite cells of sensory DRG and sympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorption and retention characteristics of selenium in dorsal root ganglion neurons

Selenium concentration in the brain tissue is far less variable than those in peripherals, such as the liver and kidneys, in rodents, when fed a selenium-deficient diet. This fact implies the importance of this element for maintaining the integrity of brain functions and the distinctive selenium metabolism and/or the regulatory mechanism in the brain. To obtain basic information concerning the homeostatically maintained selenium store in the brain, we investigated absorption and retention characteristics of selenium from selenious acid (SA) and seleno-l-methionine (SeMet) in rat dorsal root ganglion (DRG) neurons, in comparison to isolated rat hepatocytes and renal cells in vitro. When DRG neurons were cultured in an SA-free medium subsequent to an SA-supplemented one for 24 h, the DRG neurons maintained a higher selenium concentration than that before SA supplementation over a period of 96 h after removal of SA from the culture medium. The cellular glutathione peroxidase activity of the cells increased for 72 h after removal of SA from the culture medium. A similar retention characteristic of selenium was also observed for DRG neurons treated with SeMet-supplemented culture medium. Consequently, selenium from source compounds, in part, was thought to be retained in DRG neurons and then be utilized for the synthesis of selenium-containing proteins, which implied the presence of a neuron-specific selenium retention mechanism.     

Hyperpolarization-activated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are active at resting membrane potential and thus are likely to contribute to neuronal excitability. Four HCN channel subunits (HCN1–4) have previously been cloned. The aim of the current study was to investigate the immunoreactivity of HCN4 channel protein in rat trigeminal (TG) and dorsal root ganglion (DRG) sensory neurons. HCN4 was present in 9% of TG neurons and 4.7% of DRG neurons, it was distributed in a discrete population of small-diameter neurons in the TG but was located in cells of all sizes in the DRG. Approximately two thirds of HCN4-containing neurons in each ganglia were labelled with antisera raised against the 200-kDa neurofilament (NF200). The remaining HCN4-containing neurons were NF200-negative, were not labelled with antisera raised against calcitonin-gene related peptide (CGRP), and did not bind the isolectin B4 (IB4). HCN4-containing neurons made up more than half of the population of small-diameter primary afferent neurons that did not contain either NF200 or CGRP or bind IB4 in both TG and DRG. This population was not insignificant, comprising 5% of TG neurons and 2% of DRG neurons.     

Daidzein induces neuritogenesis in DRG neuronal cultures

ABSTRACT: BACKGROUND: Daidzein, a phytoestrogen found in isoflavone, is known to exert neurotrophic and neuroprotective effects on the nervous system. Using primary rat dorsal root ganglion (DRG) neuronal cultures, we have examined the potential neurite outgrowth effect of daidzein. METHODS: Dissociated dorsal root ganglia (DRG) cultures were used to study the signaling mechanism of daidzein-induced neuritogenesis by immunocytochemistry and Western blotting. RESULTS: In response to daidzein treatment, DRG neurons showed a significant increase in total neurite length and in tip number per neuron. The neuritogenic effect of daidzein was significantly hampered by specific blockers for Src, protein kinase C delta (PKCdelta) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK/ERK), but not by those for estrogen receptor (ER). Moreover, daidzein induced phosphorylation of Src, PKCdelta and ERK. The activation of PKCdelta by daidzein was attenuated in the presence of a Src kinase inhibitor, and that of ERK by daidzein was diminished in the presence of either a Src or PKCdelta inhibitor. CONCLUSION: Daidzein may stimulate neurite outgrowth of DRG neurons depending on Src kinase, PKCdelta and ERK signaling pathway.     

Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells.

Primary cultures of rat dorsal root ganglionic (DRG) cells were stained with isoform-specific antibodies against non-muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non-neuronal cells. Double staining of the cells with the anti-myosin antibodies and rhodamine-phalloidin or anti-actin antibodies indicated that MIIB co-exists, with F-actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non-neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells.