Slow chemical transmission between dorsal root ganglion neuron somata

Somatic sensory neuron somata are located within the dorsal root ganglia (DRG) and are mostly ensheathed by individual satellite glial cell sheets. It has been noted, however, that a subpopulation of these DRG somata are intimately associated, separated only by a single thin satellite glial cell membrane septum. We set out to test whether such neuron–glial cell–neuron trimers (NGlNs) are also linked functionally. The presence of NGlNs in chick DRGs was confirmed by electron microscopy. Selective satellite glial cell immunostains were identified and were used to image the inter‐neuron septa in DRG frozen sections. We used a gentle, dispase‐based enzymatic method to isolate chick and rat NGlNs in vitro for double patch clamp recordings. In the majority of pairs tested, an action potential‐like stimulus train delivered to one soma resulted in a delayed, noisy and long‐duration response in its idle partner. The response to a second stimulus train given minutes later was markedly facilitated. Both bidirectional and unidirectional transmission was observed between the paired neurons. Transmission was chemical and block by the general purinergic blocker suramin implicated ATP as a neurotransmitter. We conclude that the two neuronal somata in the NGlN can communicate by chemical transmission, which may involve a transglial, bi‐synaptic pathway. This novel soma‐to‐soma transmission reflects a novel form of processing that may play a role in sensory disorders in the DRG and interneuron communication in the central nervous system.     

Characterization of cultured dorsal root ganglion neuron P2X receptors

P2X receptors for adenosine 5′-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X_1–7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, α,β-methylene adenosine 5′-triphosphate (α,β-meATP) and 2-methyl-thioadenosine 5′-triphosphate (2-meSATP), and using the antagonists 2′,3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2′,5′ disulphonic acid (PPADS). ATP (10 μm ) evoked inward currents in ≈ 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, ≈ 4% showed a sustained response and ≈ 10% showed a combination of transient and sustained components. Rapid application of ATP, α,β-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA₅₀ values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-β,γ-methyleneadenosine 5′-triphosphate (1-β,γ-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC₅₀ = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X₃-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.     

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

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

Purinergic transmission and transglial signaling between neuron somata in the dorsal root ganglion

Most dorsal root ganglion neuronal somata (NS) are isolated from their neighbours by a satellite glial cell (SGC) sheath. However, some NS are associated in pairs, separated solely by the membrane septum of a common SGC to form a neuron–glial cell–neuron (NGlN) trimer. We reported that stimulation of one NS evokes a delayed, noisy and long‐duration inward current in both itself and its passive partner that was blocked by suramin, a general purinergic antagonist. Here we test the hypothesis that NGlN transmission involves purinergic activation of the SGC. Stimulation of the NS triggered a sustained current noise in the SGC. Block of transmission through the NGlN by reactive blue 2 or thapsigargin, a Ca²⁺ store‐depletion agent, implicated a Ca²⁺ store discharge‐linked P2Y receptor. P2Y2 was identified by simulation of the NGlN‐like transmission by puffing UTP onto the SGC and by immunocytochemical localization to the SGC membrane septum. Block of the UTP effect by BAPTA, an intracellular Ca²⁺ scavenger, supported the involvement of SGC Ca²⁺ stores in the signaling pathway. We infer that transmission through the NGlN trimer involves secretion of ATP from the NS and triggering of SGC Ca²⁺ store discharge via P2Y2 receptors. Presumably, cytoplasmic Ca²⁺ elevation leads to the release of an as‐yet unidentified second transmitter from the glial cell to complete transmission. Thus, the two NS of the NGlN trimer communicate via a ‘sandwich synapse’ transglial pathway, a novel signaling mechanism that may contribute to information transfer in other regions of the nervous system.     

Nitric oxide inhibits GABA-evoked current in dorsal root ganglion neuron via PKG-dependent pathway

gamma-Aminobutyric acid (GABA) is considered a major inhibitory neurotransmitter in the generation of presynaptic inhibition at central terminals of primary afferent fiber (PAF), while it has also been established that nitric oxide (NO) may sensitize the terminals of nocisponsive PAFs and enhance neuropeptide release, possibly via mechanisms involving the activation of a cyclic guanidine monophosphate (cGMP)-dependent PKG. The present work was undertaken to explore the modulatory effect of sodium nitroprusside (SNP), a donor of NO, on GABA-evoked current of isolated adult rat dorsal root ganglion (DRG) neurons and the intracellular mechanism involved, by means of whole-cell patch clamp recording. The results showed that 1 mM SNP reversibly inhibited the inward current evoked by 0.1 mM GABA (-1.05 +/- 0.17nA vs. -0.63 +/- 0.11nA, n = 22, p < 0.01 or 0.1 mM muscimol a specific GABA(A) receptor agonist (-1.70 +/- 0.39 nA vs. -1.01 +/- 0.24 nA, n = 6, p < 0.05), which could be cancelled by simultaneous application of 1 mM methylene blue, an inhibitor of PKG. After preapplication of SNP with increasing concentrations 0.03, 0.1, 0.3, 1, and 3 mM), SNP inhibited both 0.1 mM GABA-evoked current (IC(50) = 0.2423 mM, n = 5) and 0.1 mM muscimol-evoked current (IC(50) = 0.3255 mM, n = 3) in DRG neurons in a dose-dependent manner. Therefore, it was suggested that PKG-dependent pathway may be involved in the NO-induced inhibition of GABA(A) receptor-mediated inward current in rat DRG neurons, which may be involved in the presynaptic disinhibition of nociceptive information induced by NO under certain conditions.     

Allografted fetal dorsal root ganglion neuronal survival in the guinea pig cochlea

Neural grafting is a potential strategy to help restore auditory function following loss of spiral ganglion cells. As a first step towards the reconstruction of a neural pathway from the cochlea to the brainstem, we have examined the survival of fetal dorsal root ganglion (DRG) neurons allografted into the cochlea of adult guinea pigs. In some animals implantation of DRGs was combined with a local infusion of neurotrophic substances whereas in others auditory sensory receptors were chemically destroyed prior to DRG implantation by injection of the ototoxin neomycin into the middle ear. The results show that many transplanted DRG neurons attached close to the cochlear spiral ganglion neurons. The survival of the implant was significantly increased by treatment with neurotrophic factors, but not reduced by the absence of auditory sensory structures. This study shows that implanted sensory neurons can survive heterotopic grafting immediately adjacent to the eighth cranial nerve, thereby providing a basis for further studies of the anatomical and functional influence of neural grafts in the inner ear.     

Recognition of specific targets by cultured dorsal root ganglion neurons

We have assessed the effects of different target cell populations on axonally transported proteins by the use of compartmental cell culture systems that separate the soma from the growing axons of rat sensory neurons. The labeling of 3 rapidly transported proteins diminishes when the growing axon contacts spinal cord cells (which are normal in vivo targets), and remains unaffected by contact with fibroblasts or heart cells. Medium conditioned by spinal cord cells does not exert this effect. Thus, specific classes of cells may be distinguished as target tissue by sensory neurons in vitro. Such recognition is accompanied by specific molecular changes in axonally transported proteins.     

Transglial transmission at the dorsal root ganglion sandwich synapse: glial cell to postsynaptic neuron communication

The dorsal root ganglion (DRG) contains a subset of closely‐apposed neuronal somata (NS) separated solely by a thin satellite glial cell (SGC) membrane septum to form an NS–glial cell–NS trimer. We recently reported that stimulation of one NS with an impulse train triggers a delayed, noisy and long‐lasting response in its NS pair via a transglial signaling pathway that we term a ‘sandwich synapse’ (SS). Transmission could be unidirectional or bidirectional and facilitated in response to a second stimulus train. We have shown that in chick or rat SS the NS‐to‐SGC leg of the two‐synapse pathway is purinergic via P2Y2 receptors but the second SGC‐to‐NS synapse mechanism remained unknown. A noisy evoked current in the target neuron, a reversal potential close to 0 mV, and insensitivity to calcium scavengers or G protein block favored an ionotropic postsynaptic receptor. Selective block by D‐2‐amino‐5‐phosphonopentanoate (AP5) implicated glutamatergic transmission via N‐methyl‐d ‐aspartate receptors. This agent also blocked NS responses evoked by puff of UTP, a P2Y2 agonist, directly onto the SGC cell, confirming its action at the second synapse of the SS transmission pathway. The N‐methyl‐d ‐aspartate receptor NR2B subunit was implicated by block of transmission with ifenprodil and by its immunocytochemical localization to the NS membrane, abutting the glial septum P2Y2 receptor. Isolated DRG cell clusters exhibited daisy‐chain and branching NS–glial cell–NS contacts, suggestive of a network organization within the ganglion. The identification of the glial‐to‐neuron transmitter and receptor combination provides further support for transglial transmission and completes the DRG SS molecular transmission pathway.     

Modulation of voltage-activated Ca currents by pain-inducing agents in a dorsal root ganglion neuronal line,F-11

Whole cell currents evoked by pain-inducing agents—bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (I_Na) was 100 μA/cm² and the I_Na was abolished by 10^?6 M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient I_Ca(t) and a high threshold sustained I_Ca(I). In addition, another high threshold transient Ca current, presumably I_Ca(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of ?70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10^?6 M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10^?6 M Bk in the majority of the cells. Similarly, both 10^?6 M Cap and 10^?9 M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (G_i). © 1993 Wiley-Liss, Inc.     

Motoneurone survival and neurite regeneration requirements: the role of dorsal root ganglion cells during development

The effect of dissociated dorsal root ganglion cells on the survival of motoneurones in vitro from differently aged chick embryos has been studied. Homogeneous cultures of motoneurones were prepared from 5-8-day embryos, using a cell sorter; dorsal root ganglion cells were obtained from 8-day embryos. The survival of motoneurones from 5-day and 6-day embryos was not enhanced above controls by the presence of dorsal root ganglion cells; however, the survival of motoneurones from older embryos was greatly increased, reaching a maximum of over 80% for 8-day embryonic motoneurones. In contrast, the number of motoneurones that had regenerated neurites when co-cultured with dorsal root ganglion cells for 24 h decreased with the motoneurone age at plating, from 51% at 5 days to less than 10% for 7- and 8-day motoneurones. The survival-enhancing effects were probably mediated by cell contact between the motoneurones and processes of the dorsal root ganglion cells: conditioned media from high-density cultures of dorsal root ganglion cells could not be shown to significantly enhance the survival of motoneurones above that of control levels. The possibility that the ganglion cells exert this survival enhancing effect by depolarizing the motoneurones was examined by exposing 8-day sorted motoneurones to 47 mM potassium; this did not effect the survival of the motoneurones relative to control levels. The stage dependency of the survival of motoneurones on different neurotrophic factors and the dorsal root ganglion cell is discussed.     

The myelin-associated glycoprotein inhibitor BENZ induces outgrowth and survival of rat dorsal root ganglion cell cultures

The novel myelin-associated glycoprotein (MAG) inhibitor BENZ binds to the N-acetylneuraminic acid (Neu5Ac) portion of the N-terminal Ig-like domain of MAG. Treatment of rat dorsal root ganglion (DRG) cell cultures with BENZ-induced outgrowth of neurofilament 200-positive neurites improved survival of neurons and increased the number of GFAP-positive cells, as determined by fluorescence and confocal laser microscopy and by Western immunoblotting. Furthermore, treatment of DRG cell cultures with BENZ repressed gene and protein expression of the small GTPase RhoA but induced expression of Rho GTP-activating proteins 5 and 24, likely to counteract protein kinase A activity. Specifically, expression of inhibitors of neurite outgrowth, for example, Rock2 and PAK4, was repressed, but cofilin 1, a promoter of axonal growth, was induced. We propose that the MAG inhibitor BENZ abrogates the RhoA-ROCK-cofilin pathway to promote neurite outgrowth. Our findings require confirmation by in vivo animal studies.     

Reinnervation of denervated skeletal muscles by grafted dorsal root ganglion.

We examined whether or not the cervical dorsal root ganglion (DRG) of the rat, when isografted and connected to the distal stump of the severed common peroneal nerve, could survive, project axons to the denervated leg muscles, and exert beneficial influences to delay the degeneration of the denervated muscles. Rats in which the muscles were similarly denervated but no DRG was grafted served as the control. After a postoperative period of 72 to 286 days, histological study showed that nerve cells at the superficial part of the grafted DRG survived. Indirect electrical stimulation via the distal stump of the common peroneal nerve produced no contraction of the muscles, indicating that no functional neuromuscular contacts had been reestablished. Direct stimulation of the denervated muscles did elicit contraction, and the isometric twitch and tetanic tensions were significantly much higher in the experimental rats with a grafted DRG than in the control rats. Cholinesterase-silver staining indicated the presence of axons in the denervated muscles, but the axons did not terminate on endplates. Compared with the control muscles, the experimental muscles had significantly more axons, and had atrophied less as indicated by muscle wet weight and histological appearance. These results indicate that the sensory axons can delay the weakening and atrophy of muscles after denervation. We suggest that the sensory axons may exert certain trophic influence on the denervated muscle fibers, though the actual mechanism is unknown.     

Sequential changes of sensory neuron (fluoride-resistant) acid phosphatase in dorsal root ganglion neurons following neurectomy and rhizotomy

Five to seven days after sciatic nerve section in rats, fluoride-resistant acid phosphatase (FRAP) expression in dorsal root ganglion (drg) neurons was markedly decreased. The decrease was in contrast to increased acid phosphatase which has been reported to occur in other neurons after nerve section. FRAP expression in ganglion neurons subsequently increased 14–21 days after nerve section; this preceded the restitution of enzyme expression in the spinal cord substantia gelatinosa. FRAP expression in drg neurons was not decreased after dorsal root section.     

Neuropathy‐associated NaV1.7 variant I228M impairs integrity of dorsal root ganglion neuron axons

Small‐fiber neuropathy (SFN) is characterized by injury to small‐diameter peripheral nerve axons and intraepidermal nerve fibers (IENF). Although mechanisms underlying loss of IENF in SFN are poorly understood, available data suggest that it results from axonal degeneration and reduced regenerative capacity. Gain‐of‐function variants in sodium channel Na_V1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in ～30% of patients with idiopathic SFN. In the present study, to determine whether these channel variants can impair axonal integrity, we developed an in vitro assay of DRG neurite length, and examined the effect of 3 SFN‐associated variant Na_V1.7 channels, I228M, M932L/V991L (ML/VL), and I720K, on DRG neurites in vitro. At 3 days after culturing, DRG neurons transfected with I228M channels exhibited ～20% reduced neurite length compared to wild‐type channels; DRG neurons transfected with ML/VL and I720K variants displayed a trend toward reduced neurite length. I228M‐induced reduction in neurite length was ameliorated by the use‐dependent sodium channel blocker carbamazepine and by a blocker of reverse Na‐Ca exchange. These in vitro observations provide evidence supporting a contribution of the I228M variant Na_V1.7 channel to impaired regeneration and/or degeneration of sensory axons in idiopathic SFN, and suggest that enhanced sodium channel activity and reverse Na‐Ca exchange can contribute to a decrease in length of peripheral sensory axons. Ann Neurol 2012     

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.     

A method for unbiased and efficient estimation of number and mean volume of specified neuron subtypes in rat dorsal root ganglion

By means of unbiased stereological principles and systematic sampling techniques, the number, the mean volume, and the distribution of neuron volumes of the A- and B-cells of the dorsal root ganglion have been estimated. The number of each neuron type was estimated from the product of the volume of the ganglion, obtained with the Cavalieri principle on serial sections of the ganglion, and the numerical density, obtained with the Cavalieri principle on serial sections of the ganglion, and the numerical density, obtained with optical disectors on the same sections. The mean volume of the cell bodies of each type was estimated by applying the nucleator technique to the neurons sampled with the optical disectors. The precision of the estimate in each animal was evaluated on the basis of the variation between animals. An optimal sampling scheme is described by which estimates of the total number, the mean volume, and the distribution of cell body volumes can be obtained in about 8 hours. In the right fifth lumbar dorsal root ganglion taken from four mature, male Wistar rats, the mean total number of neurons was found to be 17,900. Of these, 28% were A-cells, with a mean cell body volume of 53,400 μm³, and 70% were B-cells, with a mean cell body volume of 8,540 μm³. There was a considerable overlap between the volume distributions of the two cell types © Wiley-Liss, Inc.     

Differentiation and maturation of zebrafish dorsal root and sympathetic ganglion neurons

The trunk neural crest of vertebrate embryos gives rise to dorsal root ganglion (DRG) sensory neurons and autonomic sympathetic neurons, among other derivatives. We have examined the development of DRG and sympathetic neurons during development in the zebrafish. We found that sensory neurons differentiate rapidly and that their overt neuronal differentiation significantly precedes that of sympathetic neurons in the trunk. Sympathetic neurons in different regions differentiate at different times. The most rostral population, which we call the cervical ganglion, differentiates several days before trunk sympathetic neurons. After undergoing overt neuronal differentiation, sympathetic neurons subsequently express the adrenergic differentiation markers dopamine beta-hydroxylase and tyrosine hydroxylase. A second population of adrenergic nonneuronal cells initially localized with cervical sympathetic neurons appears to represent adrenal chromaffin cells. In more mature fish, these cells were present in clusters within the kidneys. Individual DRG and sympathetic ganglia initially contain few neurons. However, the number of neurons in DRG and sympathetic ganglia increases continuously at least up to 4 weeks of age. Analysis of phosphohistone H3 expression and bromodeoxyuridine incorporation studies suggests that the increases in DRG and sympathetic ganglion neuronal cell number are due wholly or in part to the division of neuronal cells within the ganglia.