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.     

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.     

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.     

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.     

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.     

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.     

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.     

Urethral flow-responsive afferents in the cat sacral dorsal root ganglia

Although sensory feedback from the urethra plays an integral role in the regulation of lower urinary tract function, little is known about the properties of flow-responsive primary afferent neurons. The purpose of this study was to characterize the activity of sacral afferents that responded to fluid flow through the urethra. Single neuron action potentials were recorded extracellularly from the S1 and S2 dorsal root ganglia in eight cats anesthetized with α-chloralose. 21 of 116 cells responded to urethral flow but not to mechanical palpation of the perineum, 22 responded to both urethral flow and palpation, and 27 responded to palpation only. 34 of the 43 flow-responsive cells exhibited a firing response to 10 ml flow boluses that could be fit using a power function: FR(t) = a × (t)^b + c, where FR is firing rate, t is time, and a, b and c are constants. In all 34 cells the ‘b’ term was negative, indicating that the firing rate slowed over the time course of the urethral flow. In 16 of the 24 cells that were recorded during at least four different flow rates, a power function provided a good fit of the relationship between firing rate and flow rate: FR(flow) = k × (flow)^p + q, where k, p and q are constants. In each of these 16 cells the ‘p’ term was positive, indicating that the firing rate tended to increase with increases in flow rate. These are the first data to characterize the properties of flow-responsive afferents in the cat, and reveal properties that parallel those of other afferents.     

Epidermal growth factor receptor in adult human dorsal root ganglia

Transforming growth factor- (TGF) enhances neuronal survival and neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons. It binds a membrane protein, denominated epidermal growth factor receptor (EGFr). EGFr has been localized in developing and adult human DRG. However, it remains to be elucidated whether all DRG neurons express EGFr or whether differences exist among neuronal subtypes. This study was undertaken to investigate these topics in adult human DRG using immunoblotting, and combined immunohistochemistry and image analysis techniques. A mouse monoclonal antibody (clone F4) mapping within the intracytoplasmic domain of EGFr was used. Immunoblotting revealed two main proteins with estimated molecular masses of - 65 kDa and 170 kDa, and thus consistent with the full-length EGFr. Additional protein bands were also encountered. Light immunohistochemistry revealed specific immunoreactivity (IR) for EGFr-like proteins in most (86%) primary sensory neurons, the intensity of immunostaining being stronger in the small- and intermediate-sized ones. Furthermore, EGFr-like IR was also observed in the satellite glial cells of the ganglia as well as in the intraganglionic and dorsal root Schwann cells. Taken together, our findings demonstrate that EGFr, and other related proteins containing the epitope labeled with the antibody F4, are responsible for the EGFr IR reported in DRG. Furthermore, we demonstrated heterogeneity in the expression of EGFr-like IR in adult human primary sensory neurons, which suggests different responsiveness to their ligands.     

The transthyretin gene is expressed in human and rodent dorsal root ganglia

The transthyretin (TTR) gene is mainly expressed in the liver and choroid plexus of the brain. Most cases of familial amyloidotic polyneuropathy (FAP) are caused by TTR gene mutations, and characterized by amyloid deposition in the peripheral nervous system. We hypothesized that the TTR gene may be expressed in the peripheral nervous system. We analyzed TTR gene expression in several parts of the human, mouse and rat peripheral nervous systems using RT-PCR. To determine the sites of TTR synthesis in the dorsal root ganglia (DRG), mouse DRG were examined by in situ hybridization, laser capture microdissection and RT-PCR, and immunohistochemistry. TTR mRNA was detected in the DRG and cauda equina of humans and rodents by RT-PCR. TTR mRNA was not detected in the sural nerve, lumbar plexus or sympathetic ganglia in humans, or in the sciatic nerve in rodents. In mouse DRG, TTR mRNA was localized in the peripheral glial cells. No TTR-like immunoreactivity was observed in these tissues except for the perineurium. The TTR gene is probably expressed in the peripheral glial cells of the DRG. TTR synthesis in the DRG may be important for the involvement of the peripheral nervous system in FAP.     

Developmental changes of fucosylated glycoconjugates in rabbit dorsal root ganglia.

Developmental changes of the fucosylated glycoconjugates in the dorsal root ganglia (DRG) of the rabbit were investigated histochemically using anti-fucosyl GM1 antibody and Ulex europaeus agglutinin 1 (UEA-1) lectin. Neither anti-fucosyl GM1 antibody nor UEA-1 lectin bound to the neural tubes or to the neural crest on embryonic day 14 (E14). Anti-fucosyl GM1 antibody binds diffusely to the DRG of E25. Large neurons unreactive with anti-fucosyl GM1 antibody appeared at 1 month and increased within 6 months after birth. Schwann cells immunoreactive with anti-fucosyl GM1 antibody came to be limited to the satellite cells surrounding the positive neurons. No staining with UEA-1 lectin was observed in the DRG of E25. Some small neurons became reactive with UEA-1 lectin within 1 month and remained to be so at 6 months after birth. Schwann cells including satellite cells were unreactive with this lectin. Since fucosyl GM1 was detected in the lipid fraction of DRGs from 1-month-old and 6-month-old rabbits, fucosyl GM1 itself should be the antigen molecule recognized by the anti-fucosyl GM1 antibody. Further study is necessary to elucidate the association between these developmental changes of the fucosylated glycoconjugates in DRG and their possible functional roles.     

Neurocalcin-immunoreactive neurons in the mammalian dorsal root ganglia, including humans

Neurocalcin (NC) is a recently characterized EF-hand calcium-binding protein present in a discrete population of sensory neurons and their peripheral mechanoreceptors, but its presence in peripheral nervous system neurons other than in the rat is still unknown. The present study was designed to investigate the occurrence of NC in the dorsal root ganglia (DRG) of several mammalian species (horse, buffalo, cow, sheep, pig, dog, and rat), including humans. DRG were fixed, embedded in paraffin, and processed for immunohistochemistry using a polyclonal antibody against NC. The size of the immunoreactive neurons was measured. In all species examined, NC immunoreactivity (IR) was restricted to neurons but the percentage, as well as the size of the immunoreactive neurons, varied among different species. As a rule, small neurons (diameter <20 microm) lack NC IR. In some species (pig, dog, buffalo, cow), only the largest neurons showed IR, whereas in others (sheep, horse, rat, and humans) they covered the entire range of neuron sizes. The pattern of immunostaining was cytoplasmic, although in some species (cow and buffalo), it formed a peripheral "ring." The present results demonstrate that mammalian DRG contain a subpopulation of NC-positive neurons, which varies from one species to another. Based on the neuron size, the possible function of the NC-containing neurons is discussed.     

Somatostatin is increased in the dorsal root ganglia of adjuvant-inflamed rat

To determine whether biosynthesis of somatostatin is enhanced in the primary sensory neurons by inflammatory pain, we examined the effects of adjuvant inoculation on the content of immunoreactive somatostatin, mainly composed of somatostatin-14 and somatostatin-28, in the dorsal root ganglia and the spinal cord of the rat. The adjuvant inoculation, which produced long-lasting inflammation and hyperalgesia, increased the content of immunoreactive somatostatin, especially somatostatin-14, in the dorsal root ganglia at L4-L6 levels with no change in the dorsal and ventral horns of lumbar enlargement. Such an increase was enhanced by an intrathecal injection of colchicine (0.2 mg) that inhibits axonal flow of somatostatin. Chronic administration of the anti-inflammatory analgesic, sodium diclofenac (3 mg.kg-1.d-1), abolished an adjuvant-induced increase in the content of immunoreactive somatostatin in the dorsal root ganglia. These results suggest that the turnover (biosynthesis and axonal flow) of somatostatin in the primary sensory neurons is enhanced in the presence of persisting inflammatory pain, and support the idea that somatostatin-containing primary afferents are involved in the transmission of pain in the spinal dorsal horn.     

The relation of nucleolus diameter to cell body diameter in mammalian dorsal root ganglion cells

The present study correlates cell body and nucleolar sizes for dorsal root ganglion cells in the rat. To do this, we measured cell body areas and calculated their diameters and measured nucleolar diameters for 719 cells. These data indicate that there is a correlation in that increasing cell size is associated with increasing nucleolar size. However, there is considerable variability of cell body size for each nucleolar diameter and vice versa. Nevertheless, when nucleolar diameters are grouped, the function 1n D = 1.687 + 0.334 dr, where D is the diameter of the cell body and dr the rounded diameter of the nucleolus, produced an almost straight line. Thus this formula provides a good estimate of the relation of nucleolar and cell body sizes for dorsal root ganglion cells of the rat. In addition, estimates of the variances of cell body size at each nucleolar diameter are provided.