Neurogenesis in adult rat dorsal root ganglia

Nerve cells in mammalian species, including primary sensory neurons in the dorsal root ganglia (DRGs), are thought to be generated pre- or perinatally. The only known exceptions are olfactory receptor cells and some cortical microneurons. We now report results of experiments in which the number of neurons in the L4 and L5 DRGs of normal adult rats was counted from serial 10-micrometers paraffin sections stained with cresyl violet. Contrary to expectations, we found that there is a gradual increase in the number of DRG neurons as the animals age. The neuronal population nearly doubles over the adult life of the animal.     

Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia

Recombinant human erythropoietin (EPO) is neuroprotective in animal models of adult spinal cord injury, and reduces apoptosis in adult dorsal root ganglia after spinal nerve crush. The present work demonstrates that spinal cord and dorsal root ganglia share dynamic expression patterns of EPO and its receptor (EPOR) during development. C57Bl mice from embryonic days (E) 8 (E8) to E19 were studied. In spinal cord and dorsal root ganglia, EPOR expression in all precursor cells preceded the expression of EPO in subsets of neurons. On E11, EPO-immunoreactive spinal motoneurons and ganglionic sensory neurons resided adjacent to EPOR-expressing radial glial cells and satellite cells, respectively. From E12 onwards, EPOR-immunoreactivity decreased in radial glial cells and, transiently, in satellite cells. Simultaneously, large-scale apoptosis of motoneurons and sensory neurons started, and subsets of neurons were labelled by antibodies against EPOR. Viable neurons expressed EPO and EPOR. Up to E12.5, apoptotic cells were EPOR-immunopositive, but variably EPO-immunonegative or EPO-immunopositive. Thereafter, EPO-immunonegative and EPOR-immunopositive apoptotic cells predominated. Our findings suggest that EPO-mediated neuron-glial and, later, neuron–neuronal interactions promote the differentiation and/or the survival of subsets of neurons and glial cells in central as well as in peripheral parts of the embryonic nervous system. Correspondingly, expression of phospho-Akt-1/protein-kinase B extensively overlapped expression sites of EPO and EPOR, but was absent from apoptotic cells. Identified other sites of EPO and/or EPOR expression include radial glial cells that transform to astrocytes, cells of the floor plate and notochord as well as neural crest-derived boundary cap cells at motor exit points and cells of the primary sympathetic chain.     

Immunoelectron microscopic localization of E-cadherin in dorsal root ganglia, dorsal root and dorsal horn of postnatal mice

Summary Sensory neurons and associated glial cells are known to express the cell-cell adhesion molecule E-cadherin. The cellular and subcellular localization of this molecule in the dorsal root ganglion, dorsal root, and spinal cord of postnatal mice was studied by the pre-embedding immunoelectron microscopic labelling technique. In the dorsal root and the superficial layer of the dorsal horn, a subset of fasciculating unmyelinated axons expressed E-cadherin at their axon-axon contacts at all ages studied, and these axons were clustered together and segregated from E-cadherin-negative axons. In contrast, pre-myelinating large-diameter axons in P2 mice as well as myelinated axons in mice from P14 to adulthood were E-cadherin-negative. Glial cells also expressed E-cadherin: In the dorsal root ganglia, all of the satellite cells expressed E-cadherin at contact sites with neurons, other satellite cells, and basal lamina, at all ages studied. In dorsal roots from P14 to adulthood, myelin-forming Schwann cells expressed E-cadherin at the outer mesaxons and the contact sites with basal lamina. Non-myelin-forming Schwann cells occasionally stained for this molecule at contact sites with the plasma membrane of E-cadherin-positive axons and at other sites. These results strongly suggest that E-cadherin plays an important role in the selective fasciculation of a particular subset of unmyelinated sensory fibres, and also in glial cell contacts.     

Single-channel analysis of two types of Na+currents in rat dorsal root ganglia

The properties of voltage-gated Na⁺ channels were studied in neurones isolated from rat dorsal root ganglia using the outside-out configuration of the patch-clamp technique. Two types of single-channel currents were identified from the difference in unit amplitudes. Neither type was evoked in the medium in which extracellular Na⁺ ions were replaced by an equimolar amount of tetramethylammonium ions. The two types of single-channel currents differed in their sensitivity to tetrodotoxin (TTX). The smaller channel current was insensitive to 1 M TTX (referred to as TTX-I), while the larger channel current was blocked by 1 nM TTX (TTX-S). The unit amplitudes measured during a step depolarization to –30 mV (1.4 mM internal and 250 mM external Na⁺ concentrations) were 1.16 pA for TTX-S and 0.57 pA for TTX-I, respectively. The slope conductance measured at –30 mV was 16.3 pS for TTX-S and 8.5 pS for TTX-I. TTX-S could be activated by step depolarizations positive to –60 mV, while TTX-I could be activated at potentials positive to –40 mV. When the test pulse was preceded by a depolarizing prepulse, the prepulse positive to –50 mV preferentially inactivated TTX-S with a minimal effect on TTX-I. Activation and inactivation time courses of the averaged ensemble currents computed from TTX-S showed remarkable resemblances to the time courses of the macroscopic TTX-sensitive Na⁺ current. Similarly, the ensemble currents of TTX-I mimicked the macroscopic TTX-insensitive Na⁺ current. It was concluded that the two types of Na⁺ channels in rat dorsal root ganglia differ not only in their sensitivity to TTX, but also in their single-channel conductances.     

Somatosensory mechanisms in zebrafish lacking dorsal root ganglia

Dorsal root ganglion (DRG) sensory neurons transmit all somatosensory information from the trunk region of the body. erbb3 mutant zebrafish do not form DRG neurons because the neural crest cells that generate them migrate aberrantly. Here we report that homozygous erbb3 mutants appear to swim and feed normally, and that they survive through adulthood, despite never forming DRG neurons. The source of sensory compensation in adult erbb3 mutants remains unknown, although it may be from lateral line ganglion neuromasts which are reduced, but present, in erbb3 mutants. We also provide new information about the development of DRG neurons in wild-type juvenile zebrafish.     

Cross-excitation in dorsal root ganglia of nerve-injured and intact rats

1. Experiments based on teased fiber recording from rat sciatic nerve have shown that a small proportion of primary afferent neurons in intact dorsal root ganglia (DRGs) fire spontaneously. The prevalence of this discharge is substantially increased if the sciatic nerve has been chronically injured. 2. We now show that in most cases this ongoing DRG activity can be augmented by tetanic stimulation of the axons of neighboring neurons, where the active neuron itself has not been stimulated. In addition, some previously silent DRG neurons can be cross-excited by neighbors. This novel form of neuron-to-neuron communication is termed "DRG crossed afterdischarge." Cross-excitation never occurred at fixed latency in response to single stimulus pulses and is therefore not a case of ephaptic cross talk. 3. Crossed afterdischarge occurred only if the spontaneously active neuron and the stimulated neighbors shared the same DRG. It occurred in 83.5% of the spontaneously active neurons sampled that had myelinated (A) axons, but in only 4.4% of spontaneously active neurons with unmyelinated (C) axons. Among initially silent neurons, stimulation of neighbors evoked firing in 3.1% of A-fibers but in no C-fibers. 4. Crossed afterdischarge responses began within 500 ms of stimulation onset (with the use of 50-Hz tetani) and increased in magnitude for about the first 30 s of stimulation, declining thereafter. Intense excitations were often followed by a short period of depression until the original rate of ongoing discharge was restored. 5. The magnitude of crossed afterdischarge responses increased with increasing stimulation frequency until saturation. Minimal responses occurred with the use of tetani of as little as 1 Hz. Maximal responses occurred with the use of 100-200 Hz tetani. 6. The inclusion of C-fibers in the afferent volley produced little if any augmentation of responses. 7. Cross-excitation was demonstrated in DRGs in which many or all peripheral afferent axons were intact and continued to innervate hind limb skin. In these preparations natural cutaneous stimulation was shown to be capable of evoking crossed afterdischarge responses. The most effective stimuli were gentle or firm rubbing of the foot. Noxious pinch, heat, cold, and chemical stimulation was ineffective. 8. DRG crossed afterdischarge is a mechanism whereby sensation in response to peripheral stimulation may be distorted in time, space, and modality. Because its prevalence is much increased after axotomy, it might contribute to neuropathic sensory abnormalities, including pain, in patients with nerve injury.     

Structural basis of neuron-to-neuron cross-excitation in dorsal root ganglia

Summary Lanthanum was used as a tracer substance to determine whether small molecules in the bulk extracellular space in dorsal root ganglia have access to the narrow cleft that separates sensory neurons from their surrounding satellite cell sheath. Results showed that lanthanum is able to diffuse into this cleft, especially when the tissue is incubated with the tracer before fixation. Lanthanum gained access to the cleft at the seam where adjacent satellite cell processes meet. There appears to be preferential access in the axon hillock — initial segment region. Large diameter light neurons, which generally support fast conducting myelinated axons and carry information about non-nociceptive sensory events, proved more likely to admit lanthanum than small diameter dark neurons, which tend to have thin myelinated and unmyelinated axons and typically carry nociceptive information. Peripheral axotomy triggered a reduction in the access of lanthanum to the neuron-satellite cell cleft. These data bear on the mechanism of non-synaptic cell-to-cell cross-excitation within dorsal root ganglia, and in particular, lend support to the hypothesis that this interaction is mediated chemically rather than electrically.     

Involvement of dorsal root ganglia in Fabry''s disease.

Bouts of shooting pain along the extremities are common in the early stages of Fabry's disease. No pathological explanation has been advanced to clarify the mechanism of such pain. In the present case neuronal storage of glycolipid was confined to dorsal root ganglia neurones only. It is suggested that this may explain the shooting pain in Fabry's disease. In hereditary sensory radicular neuropathy, familial dysautonomia, and tabes dorsalis, changes in dorsal root ganglia cells cause similar clinical signs and thus it may be concluded that shooting pains in Fabry's disease may be caused by damage to dorsal root ganglia neurones.     

MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia

Previous experimental results from our laboratory demonstrated that monocyte chemoattractant protein-1 (MCP-1) depolarizes or increases the excitability of nociceptive neurons in the intact dorsal root ganglion (DRG) after a chronic compression of the DRG (CCD), an injury that upregulates neuronal expression of both MCP-1 and mRNA for its receptor CCR2. We presently explore the ionic mechanisms underlying the excitatory effects of MCP-1. MCP-1 (100 nM) was applied, after CCD, to acutely dissociated small DRG neurons with nociceptive properties. Under current clamp, the proportion of neurons depolarized was similar to that previously observed for CCD-treated neurons in the intact ganglion, although the magnitude of depolarization was greater. MCP-1 induced a decrease in rheobase by 44 +/- 10% and some cells became spontaneously active at resting potential. Action potential width at a voltage equal to 10% of the peak height was increased from 4.94 +/- 0.23 to 5.90 +/- 0.47 ms. In voltage clamp, MCP-1 induced an inward current in 27 of 50 neurons held at -60 mV, which increased with concentration over the range of 3 to 300 nM (EC(50) = 45 nM). The MCP-1-induced current was not voltage dependent and had an estimated reversal potential of -27 mV. In addition, MCP-1 inhibited a voltage-dependent, noninactivating outward current, presumably a delayed rectifier type K(+) conductance. We conclude that MCP-1 enhances excitability in CCD neurons by, at least, two mechanisms: 1) activation of a nonvoltage-dependent depolarizing current with characteristics similar to a nonselective cation conductance and 2) inhibition of a voltage-dependent outward current.     

Expression of Sema3D in subsets of neurons in the developing dorsal root ganglia of the rat

We investigated the expression of the semaphorin family member, Sema3D, in the developing dorsal root ganglia of the rat. Sema3D expression was observed in a subpopulation of dorsal root ganglion (DRG) neurons. The expression peaked at E15 and thereafter it declined. The change in Sema3D expression between E13 and E20 was analyzed by comparison to the expression of TrkA, TrkC and Neuropilin-1 by in situ hybridization. The expression pattern of Sema3D in DRG was similar to that of TrkC and was different from that of TrkA or Neuropilin-1. Double immunohistochemical analysis revealed that Sema3D expression was confined to neurons that expressed TrkC and Runx3, which mediate proprioception, but not to nociceptive neurons that express TrkA. Functionally distinct DRG neurons have different projection patterns in the spinal cord, therefore, Sema3D may regulate the axonal navigation of the large diameter DRG neurons.     

Reactive changes in dorsal roots and dorsal root ganglia after C7 dorsal rhizotomy and ventral root avulsion/replantation in rabbits

Current surgical treatment of spinal root injuries aims at reconnecting ventral roots to the spinal cord while severed dorsal roots are generally left untreated. Reactive changes in dorsal root ganglia (DRGs) and in injured dorsal roots after such complex lesions have not been analysed in detail. We studied dorsal root remnants and lesioned DRGs 6 months after C7 dorsal rhizotomy, ventral root avulsion and immediate ventral root replantation in adult rabbits. Replanted ventral roots were fixed to the spinal cord with fibrin glue only or with glue containing ciliary neurotrophic factor and/or brain-derived neurotrophic factor. Varying degrees of degeneration were observed in the deafferented dorsal spinal cord in all experimental groups. In cases with well-preserved morphology, small myelinated axons extended into central tissue protrusions at the dorsal root entry zone, suggesting sprouting of spinal neuron processes into the central dorsal root remnant. In lesioned DRGs, the density of neurons and myelinated axons was not significantly altered, but a slight decrease in the relative frequency of large neurons and an increase of small myelinated axons was noted (significant for axons). Unexpectedly, differences in the degree of these changes were found between control and neurotrophic factor-treated animals. Central axons of DRG neurons formed dorsal root stumps of considerable length which were attached to fibrous tissue surrounding the replanted ventral root. In cases where gaps were apparent in dorsal root sheaths, a subgroup of dorsal root axons entered this fibrous tissue. Continuity of sensory axons with the spinal cord was never observed. Some axons coursed ventrally in the direction of the spinal nerve. Although the animal model does not fully represent the situation in human plexus injuries, the present findings provide a basis for devising further experimental approaches in the treatment of combined motor/sensory root lesions.     

Changes in PDGF expression in spared dorsal root ganglia and associated spinal dorsal horns in cats subjected to partial dorsal root ganglionectomy

Changes in the platelet derived growth factor (PDGF) in the spared dorsal root ganglia (DRG) and associated spinal dorsal horns were evaluated in cats subjected to unilateral removal of L1-L5 and L7-S2 DRG, sparing the L6 DRG. The number of PDGF immunopositive neurons and protein expression decreased significantly in the spared DRG and associated dorsal horns of the L3 and L6 cord segments at 3 days post-operation (dpo). It bottomed to the lowest level at 7 dpo in the DRG, then returned to the control level at 14 dpo; while in the L6 dorsal horn, it rapidly increased at 7 dpo and exceeded the control level at 14 dpo. This showed a significant upregulation in the spared DRG and associated spinal dorsal horns, especially in the L6 cord segment following a transient decrease. Meanwhile, a significant upregulation of PDGF mRNA was also seen in L6 DRG and L3 and L6 dorsal horns at 3 dpo. The upregulation of the endogenous PDGF in the said structures indicated a potential role of this factor in spinal cord plasticity after partial dorsal root ganglia removal in cats.     

GABAB-mediated upregulation of the high-voltage-activated Ca2+ channels in rat dorsal root ganglia

 The mechanism underlying the enhancement of the high-voltage-activated (HVA) Ca²⁺ current (I _Ca) after application of baclofen, a GABA_B agonist, in neurones of the rat dorsal root ganglia was studied by a combined use of the nystatin perforated patch clamp recording and our rapid superfusion system. Baclofen (50 μM) decreased the peak amplitude of HVA I _Ca and slowed the onset of the current, i.e. produced a typical G-protein-mediated inhibition of I _Ca. However, when baclofen was rapidly removed from the medium, the amplitude of the current was rather augmented, exceeding the control value obtained before application of the drug. This enhancement was not due to a shift of the voltage dependence of Ca²⁺ channel activation or a change in ionic permeability to other ions. The enhancement of HVA I _Ca by baclofen was sensitive to pertussis toxin treatment. The enhancement was evident during superfusion of baclofen. Since the inhibitory effect of baclofen on HVA I _Ca was not attenuated, even after a continuous application of baclofen for 10 min, the enhancement was not due to relief from tonic G-protein-mediated inhibition of the current or a desensitization of the GABA_B receptor–effector system. An extremely prolonged time course of the enhancement of HVA I _Ca by baclofen strongly suggests an involvement of some intracellular signal transduction system. Received: 22 May 1996 / Received after revision: 17 October 1996 / Accepted: 7 January 1997     

Natural killer cell-mediated lysis of dorsal root ganglia neurons via RAE1/NKG2D interactions

Natural killer cells have been reported to be able to kill various transformed and virus-infected target cells. It was recently observed that NK cells also could kill syngeneic dorsal root ganglia (DRG) neurons by a perforin-dependent mechanism. We demonstrate here that this phenomenon does not reflect a general ability of NK cells to kill neurons in culture. While DRG neurons of the peripheral nervous system were readily killed, ventral spinal cord neurons and hippocampal neurons of the central nervous system (CNS) were resistant to lysis. The resistance to NK cell-mediated lysis of the latter neurons was not related to protection by MHC class I molecules, since similar beta(2)-microglobulin(-/-) neurons were equally resistant to lysis. While exploring other possible molecular mechanisms for the selective triggering of lysis of DRG neurons, we observed that the retinoic acid early inducible gene-1 (RAE-1), the product of which is a ligand for the NK cell-activating receptor NKG2D, was expressed at high levels in the DRG neurons. In contrast, RAE-1 was expressed only at very low levels in the resistant CNS-derived neurons. Blocking NK cells withanti-NKG2D antibodies inhibited NK cell-mediated killing of the DRG neurons. Thus, we demonstrate that NK cell-mediated lysis of DRG neurons correlates with the expression of RAE-1 and that this lysis is dependent on activation of NK cells via NKG2D. This observation demonstrates that NK cells can kill non-pathogen-infected or non-transformed syngeneic cells through activation of the NKG2D receptor.     

Chronic constriction injury induces aquaporin-2 expression in the dorsal root ganglia of rats

Aquaporins are a family of water channel proteins involved in water homeostasis in several tissues. Current knowledge of aquaporin expression in the nervous system is very limited. Therefore the first aim of this study was to assess, by immunohistochemistry and immunoblotting analysis, the presence and localization of aquaporin-2 in the spinal cord and dorsal root ganglia of naïve adult rats. In addition, we evaluated aquaporin-2 expression in response to chronic constriction injury of the sciatic nerve, a model of neuropathic pain. Our results showed that aquaporin-2 expression was not detectable either in the spinal cord or the dorsal root ganglia of naïve rats. However, we showed for the first time an increase of aquaporin-2 expression in response to chronic constriction injury treatment in small-diameter dorsal root ganglia neurons but no expression in the lumbar spinal cord. These data support the hypothesis that aquaporin-2 expression is involved in inflammatory neuropathic nerve injuries, although its precise role remains to be determined.     

Mu opioid receptor-effector coupling and trafficking in dorsal root ganglia neurons

Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely mechanism of morphine tolerance in vivo. In contrast to the muOR, the mu/partial differentialOR showed equivalent desensitization whether morphine or DAMGO treated, but internalized further with DAMGO than morphine. Such ligand-independent desensitization could be a result of the observed higher rate of synthesis and degradation of this chimeric receptor.     

Functional cross-excitation between afferent A- and C-neurons in dorsal root ganglia

Electrophysiological recordings were made in vitro from primary afferent neurons with unmyelinated axons (C-neurons) in excised rat dorsal root ganglia. Spike activity triggered in neurons with myelinated axons (A-neurons) by stimulation of the peripheral nerve or the dorsal root produced a transient depolarization in passive neighboring C-neurons that share the same ganglion. About 90% of neurons sampled responded with this "cross-depolarization". Cross-depolarization was associated with functional excitation as indicated by an increase in firing probability in response to previously subthreshold intracellular test pulses. Furthermore, it yielded a net increase of the input resistance of the affected C-neurons. We suggest that functional coupling among DRG neurons could serve a metabolic role, providing a functionally relevant feedback signal useful for controlling the excitability of nociceptive sensory endings. In addition, the results provide a novel mechanism whereby afferent nociceptors could be stimulated by activity in low-threshold mechanoreceptors, particularly in the event of nerve injury. Hence, the coupling between afferent A- and C-neurons in dorsal root ganglia provides a novel candidate mechanism for neuropathic pain.