Co-administration of Monosialoganglioside and Skeletal Muscle Cells on Dorsal Root Ganglion Neuronal Phenotypes In Vitro

The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Monosialoganglioside (GM1) has been considered to have a neurotrophic factor-like activity. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, whether target SKM cells and GM1, alone or associated, generate neuropeptide or neurofilament expression remains unclear. The aim of the present study is to investigate the effects of GM1 or/and SKM on DRG neuronal phenotypes. DRG neurons containing the neuropeptide substance P (SP) and neurofilament 200 (NF-200) were quantified using immunofluorescent labeling in cultures of DRG, which was dissected out at times before (at embryonic days 12.5, E12.5) and after (at E19.5) sensory neurons contact peripheral targets in vivo. DRG neurons were cultured in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of SP and NF-200 in E12.5 DRG cultures; (2) SKM cells promoted expression of NF-200 but not SP in E12.5 DRG cultures; (3) GM1 and target SKM cells had additive effects on expression of SP and NF-200 in E12.5 DRG cultures; and (4) SKM or/and GM1 did not have effects on expression of SP and NF-200 in E19.5 DRG cultures. These results suggested that GM1 could influence DRG, two major neuronal phenotypes, before sensory neurons contact peripheral targets in vivo. Target SKM cells could only influence neurofilament-expressed neuronal phenotype before sensory neurons contact peripheral targets in vivo. GM1 and SKM cells had the additive effects on two major DRG neuronal classes, which express neuropeptide or neurofilament when DRG cells were harvested before sensory neurons contact peripheral targets in vivo. These results offered new clues for a better understanding of the association of GM1 or/and SKM with neuronal phenotypes.     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

Formation of Neuromuscular Junctions and Synthesis of Sensory Neuropeptides in the Co-cultures of Dorsal Root Ganglion and Cardiac Myocytes

Aim The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. Methods The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). Results In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. Conclusions The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes. Zhen Liu and Huaxiang Liu contributed equally to this article.     

Distinct modulatory actions of TGF-β and LIF on neurotrophin-mediated survival of developing sensory neurons

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are important for the regulation of survival and differentiation of distinct, largely non-overlapping populations of embryonic sensory neurons. We show here that the multifunctional cytokine transforming growth factor-β (TGF-β) fails to maintain sensory neurons cultured from embryonic day (E) 8 chick dorsal root ganglia (DRG), although DRG neurons are immunoreactive for the TGF-β receptor type II, which is essential for TGF-β signaling. However, in combination with various concentrations of NT-3 and NT-4, but not NGF, TGF-β3 causes a further significant increase in neuron survival. In DRG cell cultures treated with NGF, NT-3, and NT-4, a neutralizing antibody to TGF-β decreases neuron survival suggesting that endogenous TGF-β in these cultures affects the efficacies of neurotrophins. Consistent with this notion and a modulatory role of TGF-β in neurotrophin functions is the observation that TGF-β2 and-β3 immunoreactivities and TGF-β3 mRNA are located in embryonic chick DRG in close association with neurons from E5 onwards. We also show that leukemia inhibitory factor (LIF) significantly decreases NGF-mediated DRG neuron survival. Together, these data indicate that actions and efficacies of neurotrophins are under distinct control by TGF-β and LIF in vitro, and possibly also in vivo. Special issue dedicated to Dr. Hans Thoenen.     

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development.

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.     

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons

ABSTRACT: BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. Results: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and p-CREB, and reverses cystitis-induced increases in micturition frequency. Conclusion: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.     

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.     

NT-3 and CNTF exert dose-dependent, pleiotropic effects on cells in the immature dorsal root ganglion: neuregulin-mediated proliferation of progenitor cells and neuronal differentiation

Neurons in the nascent dorsal root ganglia are born and differentiate in a complex cellular milieu composed of postmitotic neurons, and mitotically active glial and neural progenitor cells. Neurotrophic factors such as NT-3 are critically important for promoting the survival of postmitotic neurons in the DRG. However, the factors that regulate earlier events in the development of the DRG such as the mitogenesis of DRG progenitor cells and the differentiation of neurons are less defined. Here we demonstrate that both NT-3 and CNTF induce distinct dose-dependent responses on cells in the immature DRG: at low concentrations, they induce the proliferation of progenitor cells while at higher concentrations they promote neuronal differentiation. Furthermore, the mitogenic response is indirect; that is, NT-3 and CNTF first bind to nascent neurons in the DRG--which then stimulates those neurons to release mitogenic factors including neuregulin. Blockade of this endogenous neuregulin activity completely blocks the CNTF-induced proliferation and reduces about half of the NT-3-mediated proliferation. Thus, the genesis and differentiation of neurons and glia in the DRG are dependent upon reciprocal interactions among nascent neurons, glia, and mitotically active progenitor cells.     

Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review

Previously, it was believed that the lumbar intervertebral disc was innervated segmentally by dorsal root ganglion (DRG) neurons via the sinuvertebral nerves. Recently, it was demonstrated using retrograde tracing methods that the lower disc (L5-L6) is innervated predominantly by upper (L1 and L2) DRG neurons via the sympathetic trunks. Furthermore, we investigated the expression of various pain-related molecules such as calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), P2X(3) receptor and vanniloid receptor 1 (VR1) in DRG neurons innervating the disc using a combination of immunostaining with the retrograde tracing method. This review outlines the distribution and immunocytochemical characterization of DRG neurons innervating the disc. Small nociceptive DRG neurons are classified into nerve growth factor (NGF)-dependent neurons and glial cell line-derived neurotrophic factor (GDNF)-dependent neurons and they can be distinguished by their reactivity for CGRP and IB4, respectively. We found that about half of the neurons innervating the disc were CGRP-immunoreactive (-ir), whilst, only 0.6% of the DRG neurons were IB4-positive, thereby indicating that NGF-dependent neurons are the main subpopulation which transmits and modulates nociceptive information from the disc. In addition, we also demonstrated P2X(3)- and VR1-immunoreactivity in DRG neurons innervating the disc and noted that they were mainly localized in NGF-dependent neurons. It is well known that NGF has sensitizing effects on DRG neurons, with a recent study demonstratng the presence of NGF in the painful intervertebral disc. Therefore, it is suggested that NGF is involved in the generation of discogenic low back pain.     

Gestational changes in calcitonin gene-related peptide, nerve growth factor, and its receptors in rat dorsal root ganglia.

In dorsal root ganglia (DRG) cell cultures, levels of calcitonin gene-related peptide (CGRP) are increased in the presence of ovarian hormones and nerve growth factor (NGF). In addition, injection of ovariectomized rats with ovarian hormones led to an increase in levels of two NGF receptors, TrkA and p75(NTR), in DRG. Thus, we hypothesized that increased levels of ovarian hormones during pregnancy may elevate the synthesis of CGRP and NGF receptors in the DRG. DRG harvested from rats on specific days of pregnancy, on Day 2 postpartum, and after ovariectomy were subjected to radioimmunoassay, Western blot analysis, and NGF immunoassay to determine levels of CGRP, TrkA and p75(NTR), and NGF, respectively. CGRP levels in rat DRG were significantly higher during pregnancy than at Day 2 postpartum or in ovariectomized rats. Levels of both TrkA and p75(NTR) in DRG increased during pregnancy and remained elevated at Day 2 postpartum, but CGRP levels declined. Levels of NGF reached a statistically significant peak at Day 18 of gestation, and were not significantly reduced at Day 2 postpartum. Increased levels of ovarian steroid hormones during pregnancy may be involved in the synthesis of CGRP, however, the postpartum decreases in CGRP synthesis appear to be unrelated to NGF and its receptors.     

Differential Age-Dependent Trophic Responses of Nodose,Sensory, and Sympathetic Neurons to Neurotrophins and GDNF: Potencies for Neurite Extension in Explant Culture

The age-dependent trophic responses of sympathetic, sensory, and nodose neurons to the neuro-trophins NGF, BDNF, and NT-3 and to glial cell line-derived neurotrophic factor (GDNF) were examined by an explant culture system. Superior cervical ganglia (SCG), dorsal root ganglia (DRG), and nodose ganglia (NG) were removed from rat embryos (E18), neonatals ( 1 day old), young adults (3–6 months old), and aged adults (>24 months old). The ganglia were cultured with and without each neurotrophic factor; the neurite extension and neurite density were then assessed. The SCG from rats of all ages were significantly influenced by NGF, NT-3, and GDNF; the effects of NT-3 and GDNF were reduced after maturation. The DRG from embryos and neonates were influenced by all neurotrophic factors; however, the effects of BDNF and NT-3 disappeared after maturation. The GDNF showed little effect on adult DRG and no effect on aged DRG. The effect of NGF was preserved over all ages of DRG. The NG from embryonic rats were significantly responsive to BDNF and GDNF; their effects decreased in the neonatal NG, but a minimum effect remained in the aged NG. These results indicate that age-dependent profiles of trophic effects differ extensively among the lineages of the peripheral nervous system and also among the individual neurotrophic factors.     

Effect of Electroacupuncture on Neurotrophin Expression in Cat Spinal Cord after Partial Dorsal Rhizotomy

Neuroplasticity of the spinal cord following electroacupuncture (EA) has been demonstrated although little is known about the possible underlying mechanism. This study evaluated the effect of EA on expression of neurotrophins in the lamina II of the spinal cord, in cats subjected to dorsal rhizotomy. Cats received bilateral removal of L1–L5 and L7–S2 dorsal root ganglia (DRG, L6 DRG spared) and unilateral EA. They were sacrificed 7 days after surgery, and the L6 spinal segment removed and processed by immunohistochemistry and in situ hybridization histochemistry, to demonstrate the expression of neurotrophins. Significantly greater numbers of nerve growth factor (NGF) and neurotrophin-3 (NT-3) positive neurons, brain-derived neurotrophic factor (BDNF) immunoreactive varicosities and NT-3 positive neurons and glial cells were observed in lamina II on the acupunctured (left) side, compared to the non-acupunctured, contralateral side. Greater number of neurons expressing NGF mRNA was also observed on the acupunctured side. No signal for mRNA to BDNF and NT-3 was detected. The above findings demonstrate that EA can increase the expression of endogenous NGF at both the mRNA and protein level, and BDNF and NT-3 at the protein level. It is postulated that EA may promote the plasticity of the spinal cord by inducing increased expression of neurotrophins.     

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3

INTRODUCTION: Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined. OBJECTIVE: The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3. METHODS: Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA. RESULTS: Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3. CONCLUSIONS: Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.     

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X_{1 –7}). P2X₃ homomultimer and P2X_2/3 heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X₅ receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X₅ receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X₅ receptors in cat. In mouse DRG, the P2X₅ receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X₅ receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X₅ receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X₅-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X₅-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X₅ receptors were coexpressed with NF-200 or with NOS. Only a few P2X₅-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X₅ receptors.     

High levels of mRNA coding for substance P, somatostatin and alpha-tubulin are expressed by rat and rabbit dorsal root ganglia neurons

Oligonucleotide probes complementary to alpha-tubulin, preprotachykinin A (PPT A), preprosomatostatin (PPSOM), and preproarginine-vasopressin (PPAVP) mRNA were hybridized to sections of rat and rabbit brain and dorsal root ganglia (DRG) at all spinal levels. Approximately 100% of the DRG neurons in the rat and rabbit express alpha-tubulin mRNA, 20-30% express PPT A mRNA and 5-17% express PPSOM mRNA. Whereas neurons which express PPSOM mRNA are of relative uniform size, the neurons which express PPT A mRNA segregate into two broad groups. One group is composed of smaller neurons (200-2,000 microns 2) which contain an extremely dense concentration of PPT A mRNA. The second group is composed of larger neurons (2,000-3,500 microns 2) which contain a moderate concentration of PPT A mRNA. PPAVP mRNA is present in very high concentrations in the paraventricular and supraoptic nucleus of the rat hypothalamus but is not detected in any DRG neurons. In both the rat and the rabbit the density of PPT A and PPSOM mRNA is high in individual DRG neurons in comparison to PPT A and PPSOM mRNA levels contained in most forebrain neurons. These results suggest that although the level of neuropeptide present in DRG neurons is relatively low in comparison to other brain areas, the rate of sensory neuropeptide synthesis and turnover, as reflected by mRNA content, is extremely high.