Developmental regulation of notochord-derived repulsion for dorsal root ganglion axons

During the initial stages of development, the notochord provides repulsive signals for dorsal root ganglion (DRG) axons via semaphorin 3A/neuropilin-1, axonin-1/SC2, and other unknown repulsive molecules. The notochord is known to produce aggrecan, one of the chondroitin sulfate proteoglycans (CSPGs). We report here that adding aggrecan to the culture medium cannot only induce DRG growth cone collapse, but also inhibit DRG axonal growth. Using cocultures composed of tissues derived from chick embryos or neuropilin-1-deficient mice treated with chondroitinase ABC, we show the direct evidence that CSPGs are involved in notochord-derived repulsion for DRG axons. At later developmental stages, CSPGs are involved in perinotochordal sheath-derived axon repulsion, but not in notochord core-derived repulsion. We further demonstrate that TAG-1/axonin-1/SC2 is not involved in mediating repulsive activities by CSPGs, but is required for notochord core-derived axon repulsion. Thus, notochord-derived multiple axon repulsions act in a spatiotemporal-specific manner to shape the initial trajectories of DRG axons.     

Axonal regeneration in bifurcating axons of rat dorsal root ganglion cells

The rate of axonal regeneration in rats was determined by using fast axonal transport, and the following findings are reported. (i) Rate of axonal regeneration in the central branch was lower than that in the peripheral one, and this asymmetry persisted throughout the postnatal life examined. (ii) Rate of axonal regeneration slowed with age in both the central and the peripheral branches. (iii) Initial delay increased with age.     

The ratio of dorsal root ganglion cells to dorsal root axons in sacral segments of the cat

Processes of dorsal root ganglion cells are depicted as being unbranched until they reach the spinal cord or periphery. This is an important concept because, for example, branching of these processes might be a basis for referred pain. Recently several studies indicate that axons of rat dorsal root ganglion cells branch in or near the ganglion. The present study extends this work by showing that there are approximately 1.4 dorsal root axons for each dorsal root ganglion cell in sacral segments of the adult cat, and these data are interpreted as indicating that many dorsal root axons in this animal also branch. Thus this study provides further evidence to indicate that a revision of our ideas about the organization of primary sensory cells is desirable. In addition, this study provides data to indicate that the numbers of both dorsal root ganglion cells and dorsal root axons differ on the right as compared to the left side of the same segment.     

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.     

Numbers of rat dorsal root axons and ganglion cells during postnatal development

The present study demonstrates that T4 and S2 rat dorsal root axons decrease significantly from birth to adulthood with almost all of the decrease occurring in the first two weeks of life. Dorsal root ganglion cell numbers do not change during this time period. This is thus an example of postnatal axon elimination not associated with death of the cells that give rise to the axons. Presumably this regressive process is important in the formation of the normal adult nervous system. In addition, these findings raise the possibility that certain types of neonatal denervation may increase adult axon numbers by stopping a regressive process, the loss of axons, rather than initiating a progressive process, the formation of new axons.     

Number of dorsal root ganglion neurons and axons in cats of different ages

Neurons and axons were counted in normal L7 dorsal root ganglia (DRG) and dorsal roots of cats from 2 months to 11 years of age. Over this time period no change in neuronal but a slight increase in axonal numbers were observed. These findings indicate that in contrast to previous reports on male rat lumbar ganglia, neurogenesis in cat DRG does not continue into adulthood.     

A conditioning lesion of the peripheral axons of dorsal root ganglion cells accelerates regeneration of only their peripheral axons

Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromatolysis and changes in protein synthesis in DRG cells. We investigated whether a stimulus produced by peripheral branch axotomy would affect the regenerative properties of both the central and peripheral axon of the DRG cell equally. To examine this question, a conditioning crush lesion was made distally on the sciatic nerve 2 weeks prior to a testing lesion of either the dorsal root or peripheral branch axon near the DRG. Fast axonal transport of radioactive proteins was used to assess regeneration of DRG axons. In the adult rat, leading peripheral branch axons normally regenerate at a rate of 4.4 mm/day. If a conditioning lesion of the sciatic nerve is made 2 weeks before the test lesion, the rate of peripheral branch axonal regeneration increases by 25% to 5.5 mm/day. This effect is not limited to the fastest growing axons in the nerve since a population of more slowly growing axons also exhibits accelerated outgrowth in response to a prior peripheral axotomy. In contrast to this, the fastest growing central branch axons of DRG cells, which normally regenerate at a rate of 2.5 mm/day, are not significantly affected by a prior peripheral axotomy. A population of more slowly growing axons in the dorsal root also does not exhibit accelerated outgrowth in response to a peripheral conditioning lesion. The results of these experiments indicate that changes in the DRG neuron's metabolism induced by prior axotomy of its peripheral axon do not affect the regenerative properties of both axons equally. This raises the possibility that accelerated axonal outgrowth in only one axonal branch results from a differentially regulated supply of proteins to the two axons by the DRG cell body.     

Anatomical studies of dorsal column axons and dorsal root ganglion cells after spinal cord injury in the newborn rat

The response of dorsal column axons was studied after neonatal spinal overhemisection injury (right hemicord and left doral funiculus). Rat pups (N = 11) received this spinal lesion at the C2 level within 30 hours after birth. The cauda equina was exposed 3 months later in one group of chronic operates (N = 5) and in a group of normal adults (N = 2), and all spinal roots from L5 caudally were cut bilaterally; 4 days later the spinal cord and medulla were processed for Fink-Heimer impregnation of degenerating axons and terminals. In a second group of chronic operates (N = 6) and normal adult controls (N = 4) the left sciatic nerve was injected with a cholera toxin-HRP conjugate (C-HRP), followed by a 2-3 day transganglionic transport period, and then the spinal cord and medulla were processed with tetramethylbenzidine histochemistry. Both control groups have a consistent dense projection in topographically adjacent regions of the dorsal funiculus and gracile nucleus. However, there is no sign of axonal growth around the lesion in either group of chronic experimental operates. Instead, there is a decreased density of projection within the dorsal funiculus near the lesion site. Many remaining C-HRP labeled axons in the experimental operates have abnormal, thick varicosities and swollen axonal endings (5-10 microns x 10-30 microns) within the dorsal funiculus through several spinal segments caudal to the lesion. Ultrastructural analysis of the dorsal funiculus in three other chronic experimental operates reveals the presence of numerous vesicle filled axonal profiles and reactive endings which appear similar to the C-HRP labeled structures. Transganglionic labeling after C-HRP sciatic nerve injections (N = 4) and retrograde labeling of L4, L5 dorsal root ganglion neurons after fast blue injections of the gracile nucleus (N = 6) both suggest that all dorsal column axons project to the gracile nucleus in the newborn rat. Dorsal root ganglion (DRG) cell survival following the neonatal overhemisection injury was also examined in the L4 and L5 DRG. DRG neurons that project to the gracile nucleus were prelabeled by injecting fast blue into this nucleus at birth two days prior to the cervical overhemisection spinal injury. Both normal littermates (N = 9) and spinally injured animals (N = 12) were examined after postinjection survival periods of 10 or 22 days.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Inhibition of acid‐sensing ion channels by levo‐tetrahydropalmatine in rat dorsal root ganglion neurons

Levo‐tetrahydropalmatine (l‐THP), a main bioactive Chinese herbal constituent from the genera Stephania and Corydalis, has been in use in clinical practice for years in China as a traditional analgesic agent. However, the mechanism underlying the analgesic action of l‐THP is poorly understood. This study shows that l‐THP can exert an inhibitory effect on the functional activity of native acid‐sensing ion channels (ASICs), which are believed to mediate pain caused by extracellular acidification. l‐THP dose dependently decreased the amplitude of proton‐gated currents mediated by ASICs in rat dorsal root ganglion (DRG) neurons. l‐THP shifted the proton concentration–response curve downward, with a decrease of 40.93% ± 8.45% in the maximum current response to protons, with no significant change in the pH_0.5 value. Moreover, l‐THP can alter the membrane excitability of rat DRG neurons to acid stimuli. It significantly decreased the number of action potentials and the amplitude of the depolarization induced by an extracellular pH drop. Finally, peripherally administered l‐THP inhibited the nociceptive response to intraplantar injection of acetic acid in rats. These results indicate that l‐THP can inhibit the functional activity of ASICs in dissociated primary sensory neurons and relieve acidosis‐evoked pain in vivo, which for the first time provides a novel peripheral mechanism underlying the analgesic action of l‐THP. © 2014 Wiley Periodicals, Inc.     

Observations on the morphology of axons and somata of slowly conducting dorsal root ganglion cells in the cat

In anaesthetized cats, the somata of cervical dorsal root ganglion (DRG) cells were impaled with glass micropipettes containing horseradish peroxidase. Slowly conducting afferent units were first classified by the conduction velocity of the peripheral axon (group IV: less than 2.5 m/s, group III: 2.5-30 m/s) and then injected iontophoretically with horseradish peroxidase in order to visualize the perikaryon and the axons close to the ganglion. All units classified as group IV had non-myelinated peripheral and central axons. DRG cells classified as group III were morphologically heterogeneous. Units conducting in the lower group III range had non-myelinated axons on both sides of the bifurcation; in those having an intermediate conduction velocity only the peripheral axon was myelinated, and in the fastest conducting group III units both the peripheral and central axons were myelinated. One out of the 36 stained cells gave rise to 3 processes, namely 1 central axon and 2 peripheral branches. The latter ones left the ganglion at its distal pole. Within the ganglion a significant tapering of the peripheral axon of group IV afferent units was observed. In the frequency distribution histogram of cell sizes, the cross-sectional areas of group IV somata were not restricted to the lower extreme of the distribution, but showed a great overlap with the somata of group III and even group II and I units.     

Age-dependent control of dorsal root ganglion neuron survival by macromolecular and low-molecular-weight trophic agents and substratum-bound laminins

Chick embryo dorsal root ganglion (DRG) neurons can be supported in vitro by nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). Pyruvate is also required for survival of neurons from embryonic day 8 (E8) chick ciliary ganglia and from several chick and rat embryonic central nervous system sources. Here we have examined the survival requirements of chick DRG neurons between E6.5 and E15. These DRG neurons, initially dependent only on NGF, become dependent also on CNTF and later on increasingly independent from both factors. Pyruvate nearly doubles neuronal survival at all ages under all conditions. The pyruvate concentration permitting this additional survival was reduced two-fold with serine present. In the presence of polyornithine-bound laminins, nearly all seeded neurons were rescued by pyruvate plus NGF (E8 on), or pyruvate plus CNTF (E10 on), or pyruvate without trophic factors (E15). The same maximal survival was achieved without pyruvate by supplying E10 or older neurons with both NGF and CNTF. Unmodified polyornithine substrata yielded about one-half this number of surviving neurons.     

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.     

Toll‐like receptor 4 deficiency impairs microglial phagocytosis of degenerating axons

Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial‐axon co‐culture to explore the role of Toll‐like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type‐1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4‐dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4‐mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration. GLIA 2014;62:1982–1991     

Regeneration of transected dorsal root ganglion cell axons into the spinal cord in adult frogs (Xenopus laevis)

The extent of regeneration of the central axonal processes of dorsal root ganglion cells was determined using anterograde horseradish peroxidase histochemistry at 1–12 weeks after dorsal root transection in adult frogs. At 4, 8 and 12 weeks axons were found to have regenerated across the dorsal root entry zone and into the spinal cord.