The development of an identified subpopulation of avian sensory neurons is regulated by interaction with the periphery

The monoclonal antibody, SN1, binds to the cell surface of a subpopulation of avian sensory neurons. Dorsal root ganglia (DRG) that innervate different peripheral fields contain significantly different proportions of SN1(+) neurons. Moreover, the developmental time of appearance of these neurons suggests that they normally express SN1 immuno-reactivity only after they have made contact with their normal peripheral targets (Marusich et al., 1986). In the present paper, we test the hypothesis that the proportion of SN1(+) neurons within DRG is regulated by interactions between the developing neurons and their peripheral targets. Thus, immature DRG explanted into a defined medium show an age-dependent increase in the proportion of cells able to become SN1(+), but fail to show the large increase in number of immunoreactive neurons that occurs in vivo. Moreover, unilateral wing bud extirpations performed at E3 (prior to wing innervation) resulted in a dramatic selective decrease in the number of SN1(+) neurons within DRG that normally innervate the wing. These results support the hypothesis that peripheral targets regulate the appearance of SN1(+) neurons.     

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal trasnport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine wheter the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se. © 1993 John Wiley & Sons, Inc.     

Structure and expression of differentiation antigens on functional subclasses of primary sensory neurons

Subpopulations of dorsal root ganglion neurons can be distinguished on the basis of their peripheral receptive properties, spinal terminal arbors and neuropeptide content. We have used monoclonal antibodies (MAbs) to define antigenic determinants on functional populations of DRG neurons projecting to the superficial dorsal horn of the spinal cord. Three MAbs recognize defined carbohydrate epitopes associated with lacto- and globo-series glycolipids that constitute the stage-specific embryonic antigens (SSEAs) 1, 3 and 4. SSEA-3 and SSEA-4 are present in the cytoplasm of about 10% of DRG neurons in adult rat. These neurons are distinct from those that contain substance P, somatostatin or the fluoride-resistant acid phosphatase enzyme, FRAP. SSEA-1 is present in a small percentage of DRG neurons. SSEAs are present on the surface of DRG neurons maintained in dissociated cell culture: 6% are SSEA-1+, 7% are SSEA-3+ and 10-15% are SSEA-4+. MAbs LD2, KH10, TC6 and TD10 identify epitopes expressed coincidently in 25% of small DRG neurons that project to lamina II of the dorsal horn. All somatostatin- but less than 1% of substance P-immunoreactive DRG neurons express these antigens. MAb LA4 labels a distinct population of small DRG neurons that also projects to lamina II. There is extensive overlap between LA4+ neurons and those that contain FRAP. Antigens recognized by these MAbs are expressed on the surface of 10-20% of DRG neurons in culture. Preliminary biochemical studies suggest that these antigens may be glycolipids. Molecules bearing carbohydrate differentiation antigens may be involved in the development and specification of sensory connections in the dorsal horn of the spinal cord.     

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ～25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.     

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).     

Caspase-2 Is Upregulated after Sciatic Nerve Transection and Its Inhibition Protects Dorsal Root Ganglion Neurons from Apoptosis after Serum Withdrawal

Sciatic nerve (SN) transection-induced apoptosis of dorsal root ganglion neurons (DRGN) is one factor determining the efficacy of peripheral axonal regeneration and the return of sensation. Here, we tested the hypothesis that caspase-2 (CASP2) orchestrates apoptosis of axotomised DRGN both in vivo and in vitro by disrupting the local neurotrophic supply to DRGN. We observed significantly elevated levels of cleaved CASP2 (C-CASP2), compared to cleaved caspase-3 (C-CASP3), within TUNEL+DRGN and DRG glia (satellite and Schwann cells) after SN transection. A serum withdrawal cell culture model, which induced 40% apoptotic death in DRGN and 60% in glia, was used to model DRGN loss after neurotrophic factor withdrawal. Elevated C-CASP2 and TUNEL were observed in both DRGN and DRG glia, with C-CASP2 localisation shifting from the cytosol to the nucleus, a required step for induction of direct CASP2-mediated apoptosis. Furthermore, siRNA-mediated downregulation of CASP2 protected 50% of DRGN from apoptosis after serum withdrawal, while downregulation of CASP3 had no effect on DRGN or DRG glia survival. We conclude that CASP2 orchestrates the death of SN-axotomised DRGN directly and also indirectly through loss of DRG glia and their local neurotrophic factor support. Accordingly, inhibiting CASP2 expression is a potential therapy for improving both the SN regeneration response and peripheral sensory recovery.     

The Plasticity of the DRG Neurons Belonging to Different Subpopulations After Dorsal Rhizotomy

SUMMARY 1. The plasticity of sensory neurons following the injury to their axons is very important for prognosis of recovery of afferent fibers with different modality. It is evident that the response of dorsal root ganglion (DRG) neurons after peripheral axotomy is different depending on the deficiency in neurotrophic factors from peripheral region. The loss of cells appears earlier and is more severe in B-cells (small, dark cells with unmyelinated axons) than in A-cells (large, light cells with myelinated axons).2. We studied using immunohistochemical methods the response of DRG neurons to dorsal rhizotomy and combined injury of central and peripheral neuronal processes. A quantitative analysis of DRG neurons tagged by the selective markers isolectin B4 (IB4) and the heavy molecular component of the neurofilament triplet (NF200) antibody, selective for subpopulations of small and large/medium DRG neurons, respectively, was performed after dorsal rhizotomy, peripheral axotomy, and their combination.3. The number of NF200⁺-neurons is reduced substantially after both dorsal rhizotomy and peripheral axotomy, while the decrease of IB4⁺-neurons is observed only in combined injury, i.e., dorsal rhizotomy accompanied with sciatic nerve injury.4. Our results show that distinct subpopulations of DRG neurons respond differently to the injury of their central processes. The number of NF200⁺-neurons decreases to greater degree following dorsal rhizotomy in comparison to IB4⁺-neurons.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

TNBS ileitis evokes hyperexcitability and changes in ionic membrane properties of nociceptive DRG neurons

This study examines whether intestinal inflammation leads to changes in the properties of ion channels in dorsal root ganglia (DRG) neurons. Ileitis was induced by injection of trinitrobenzene sulfonic acid (TNBS), and DRG neurons innervating the ileum were labeled using fast blue. Intracellular recording techniques were used to measure electrophysiological properties of acutely dissociated neurons 12-24 h after dissection. Nociceptive neurons were identified by sensitivity to capsaicin, tetrodotoxin resistance, and size (<30 microm). The action potential threshold in neurons from TNBS-treated animals was reduced by >70% compared with controls (P < 0.001), but the resting membrane potential was unchanged. Cell diameter, input resistance (67%), and action potential upstroke velocity (22%) increased in the TNBS group (P < 0.05). The number of action potentials discharged increased in the TNBS group (P < 0.001), whereas application of 4-aminopyridine to control cells mimicked this effect. This study demonstrates that ileitis induces hyperexcitability in nociceptive DRG neurons and changes in the properties of Na(+) and K(+) channels at the soma, which persist after removal from the inflamed environment.     

Runx1 selectively regulates cell fate specification and axonal projections of dorsal root ganglion neurons

Runx1-deficient mice die around embryonic day 11.5 due to impaired hematopoiesis. This early death prevents the analysis of the role of Runx1 in the development of sensory ganglia. To overcome the early embryonic lethality, we adopted a new approach to utilize transgenic Runx1-deficient mice in which hematopoietic cells are selectively rescued by Runx1 expression under the control of GATA-1 promoter. In Runx1-deficient mice, the total number of dorsal root ganglion (DRG) neurons was increased, probably because of an increased proliferative activity of DRG progenitor cells and decreased apoptosis. In the mutant DRG, TrkA-positive neurons and peptidergic neurons were increased, while c-ret-positive neurons were decreased. Axonal projections were also altered, in that both central and peripheral projections of CGRP-positive axons were increased. In the dorsal horn of the spinal cord, projections of CGRP-positive axons expanded to the deeper layer, IIi, from the normal terminal area, I/IIo. Our results suggest that Runx1 is involved in the cell fate specification of cutaneous neurons, as well as their projections to central and peripheral targets.     

Immunohistochemical detection of islet-1 and neuronal nitric oxide synthase in the dorsal root ganglia (DRG) of sheep fetuses during gestation.

This study first investigated the ontogeny of Islet-1 and neuronal nitric oxide synthase (nNOS) expression and their co-localization in the DRG of sheep fetuses during gestation by immunohistochemistry (IHC). The results showed that Islet-1 and nNOS were located in the nuclei and cytoplasm of DRG neurons, respectively. The relative percentages of Islet-1-immunopositive (Islet-1(+)) neurons accounting for the total DRG neurons were 90%, 79%, 66%, and 53% at days 60, 90, and 120 of gestation and postnatally, respectively. The percentage of nNOS-immunopositive (nNOS(+)) neurons was 94% at day 60 and declined to approximately 30% at day 90, with no obvious further change until the postnatal period. Dual IHC showed that approximately 69% Islet-1(+) neurons express nNOS at day 60 of gestation. This proportion declined to approximately 24% at day 90, after which there was no significant change until birth. We also observed that most Islet-1(+) and nNOS(+) neurons belonged to small and medium-sized DRG neurons from day 90 of gestation to the postnatal period. These results suggest that both Islet-1 and nNOS are important for the differentiation and maintenance of some specific phenotypes of DRG neurons during late gestation of sheep fetuses, although the related mechanisms need to be further elucidated.     

Promotion of survival and neurite outgrowth of cultured peripheral neurons by exogenous lipids and detergents

Gangliosides, in particular the monosialoglycosphingolipids Gtet 1 (GM1), have previously been implicated in the mediation of neuronal rescue and restitutional axonal growth, both in vitro and subsequent to brain and peripheral nerve lesions. In the present study it is shown that the bis-sialosyl gangliosides Gtet2b and Gtet3b, but not the gangliosides Gtet2a and Gtet1, promote the survival of dissociated dorsal root ganglion (DRG) neurons cultured from Embryonic Day (E) 8 chicks (DRG8) almost to the same extent as nerve growth factor (NGF). Ciliary ganglion (CG) neurons from E8 chicks (CG8) and DRG10 neurons were virtually not supported suggesting considerable specificity in terms of neuronal targets and developmental stages being addressed. Moreover, a variety of other lipids including cerebroside (Cb), dipalmitoylphosphatidylcholine (DPPC) and -serine (DPPS), sulfatide (Sf), and sphingomyelin (Sm) were tested for putative survival promoting activity toward chick CG, DRG, and lumbar sympathetic ganglion (SG11) neurons. At the highest concentration employed (2.5 x 10(-5) M), Sm, DPPC, and DPPS maintained between 45 and 65% of the plateau survival with CG8 (maximally supported by ciliary neuronotrophic factor (CNTF], DRG8, and DRG10 neurons, and 30 to 40% with SG11 neurons. Cb supported CG8 neurons at about 55% of the plateau value achieved with CNTF, but had hardly any effect on the other neuron populations tested. Control experiments using highly enriched neurons and serum-free conditions assured that the effects were unlikely to be mediated by serum components or nonneuronal cells. A variety of detergents, in particular Triton X-100, also promoted the survival of CG8 and DRG10 neurons. Ganglioside Gtet1, Sm, and Triton X-100 shifted the NGF titration curve for DRG10 neurons between 6- and 15-fold in a dose-dependent manner suggesting synergisms between NGF and lipids for neuronal maintenance. These results document the neuronotrophic potency of certain gangliosides, a heterogeneous group of structurally unrelated lipids, and detergents. The mechanisms by which these agents modulate neuronal survival still await clarification.     

AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X(3), respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X(3)- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.