Nociceptor subtypes are born continuously over DRG development.

Sensory neurogenesis in the dorsal root ganglion (DRG) occurs in two waves of differentiation with larger, myelinated proprioceptive and low-threshold mechanoreceptor (LTMR) neurons differentiating before smaller, unmyelinated (C) nociceptive neurons. This temporal difference was established from early birthdating studies based on DRG soma cell size. However, distinctions in birthdates between molecular subtypes of sensory neurons, particularly nociceptors, is unknown. Here, we assess the birthdate of lumbar DRG neurons in mice using a thymidine analog, EdU, to label developing neurons exiting mitosis combined with co-labeling of known sensory neuron markers. We find that different nociceptor subtypes are born on similar timescales, with continuous births between E9.5 to E13.5, and peak births from E10.5 to E11.5. Notably, we find that thinly myelinated Aδ-fiber nociceptors and peptidergic C-fibers are born more broadly between E10.5 and E11.5 than previously thought and that non-peptidergic C-fibers and C-LTMRs are born with a peak birth date of E11.5. Moreover, we find that the percentages of nociceptor subtypes born at a particular timepoint are the same for any given nociceptor cell type marker, indicating that intrinsic or extrinsic influences on cell type diversity are occurring similarly across developmental time. Overall, the patterns of birth still fit within the classical "two wave" description, as touch and proprioceptive fibers are born primarily at E10.5, but suggest that nociceptors have a slightly broader wave of birthdates with different nociceptor subtypes continually differentiating throughout sensory neurogenesis irrespective of myelination.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Pioneer Axons Utilize a Dcc Signaling-Mediated Invasion Brake to Precisely Complete Their Pathfinding Odyssey.

Axons navigate through the embryo to construct a functional nervous system. A missing part of the axon navigation puzzle is how a single axon traverses distinct anatomic choice points through its navigation. The dorsal root ganglia (DRG) neurons experience such choice points. First, they navigate to the dorsal root entry zone (DREZ), then halt navigation in the peripheral nervous system to invade the spinal cord, and then reinitiate navigation inside the CNS. Here, we used time-lapse super-resolution imaging in zebrafish DRG pioneer neurons to investigate how embryonic axons control their cytoskeleton to navigate to and invade at the correct anatomic position. We found that invadopodia components form in the growth cone even during filopodia-based navigation, but only stabilize when the axon is at the spinal cord entry location. Further, we show that intermediate levels of DCC and cAMP, as well as Rac1 activation, subsequently engage an axon invasion brake. Our results indicate that actin-based invadopodia components form in the growth cone and disruption of the invasion brake causes axon entry defects and results in failed behavioral responses, thereby demonstrating the importance of regulating distinct actin populations during navigational challenges.SIGNIFICANCE STATEMENT Correct spatiotemporal navigation of neuronal growth cones is dependent on extracellular navigational cues and growth cone dynamics. Here, we link dcc-mediated signaling to actin-based invadopodia and filopodia dynamics during pathfinding and entry into the spinal cord using an in vivo model of dorsal root ganglia (DRG) sensory axons. We reveal a molecularly-controlled brake on invadopodia stabilization until the sensory neuron growth cone is present at the dorsal root entry zone (DREZ), which is ultimately essential for growth cone entry into the spinal cord and behavioral response.

Isolation and Culture of Dorsal Root Ganglia (DRG) from Rodents.

Preparations of peripheral sensory neurons from rodents are essential for studying the molecular mechanism of neuronal survival and physiology. Although, isolating and culturing these neurons proves difficult, often these preparations are contaminated with nonneuronal proliferating cells. Here, we describe an isolation method using a Percoll gradient and an antimitotic reagent to significantly reduce the nonneuronal cell contamination while maintaining the integrity of the rodent sensory dorsal root ganglia (DRG) neurons.

Innervation of Meissner's corpuscles and Merkel -cells by transplantation of embryonic dorsal root ganglion cells after peripheral nerve section in rats.

Transplantation of embryonic motor neurons has been shown to improve motor neuron survival and innervation of neuromuscular junctions in peripheral nerves. However, there have been no reports regarding transplantation of sensory neurons and innervation of sensory receptors. Therefore, we hypothesized that the transplantation of embryonic sensory neurons may improve sensory neurons in the skin and innervate Merkel cells and Meissner's corpuscles. We obtained sensory neurons from dorsal root ganglia of 14-day rat embryos. We generated a rat model of Wallerian-degeneration by performing sciatic nerve transection and waiting for one week after. Six months after cell transplantation, we performed histological and electrophysiological examinations in naïve control, surgical control, and cell transplantation groups. The number of nerve fibers in the papillary dermis and epidermal-dermal interface was significantly greater in the cell transplantation than in the surgical control group. The percent of Merkel cells with nerve terminals, as well as the average number of Meissner corpuscles with nerve terminals, were higher in the cell transplantation than in the surgical control group, but differences were not significant between the two groups. Moreover, the amplitude and latency of sensory conduction velocity were evoked in rats of the cell transplantation group. We demonstrated that the transplantation of embryonic dorsal root ganglion cells improved sensory nerve fiber number and innervation of Merkel cells and Meissner's corpuscles in peripheral nerves.

Expression of Connexins 37, 43 and 45 in Developing Human Spinal Cord and Ganglia.

Direct intercellular communication via gap junctions has an important role in the development of the nervous system, ranging from cell migration and neuronal differentiation to the formation of neuronal activity patterns. This study characterized and compared the specific spatio-temporal expression patterns of connexins (Cxs) 37, 43 and 45 during early human developmental stages (since the 5th until the 10th developmental week) in the spinal cord (SC) and dorsal root ganglia (DRG) using double immunofluorescence and transmission electron microscopy. We found the expression of all three investigated Cxs during early human development in all the areas of interest, in the SC, DRG, developing paravertebral ganglia of the sympathetic trunk, notochord and all three meningeal layers, with predominant expression of Cx37. Comparing the expression of different Cxs between distinct developmental periods, we did not find significant differences. Specific spatio-temporal pattern of Cxs expression might reflect their relevance in the development of all areas of interest via cellular interconnectivity and synchronization during the late embryonic and early fetal period of human development.

Synergistic effect of CNTF and GDNF on directed neurite growth in chick embryo dorsal root ganglia.

It is often critical to improve the limited regenerative capacity of the peripheral nerves and direct neural growth towards specific targets, such as surgically implanted bioengineered constructs. One approach to accomplish this goal is to use extrinsic neurotrophic factors. The candidate factors first need to be identified and characterized in in vitro tests for their ability to direct the neurite growth. Here, we present a simple guidance assay that allows to assess the chemotactic effect of signaling molecules on the growth of neuronal processes from dorsal root ganglia (DRG) using only standard tissue culture materials. We used this technique to quantitatively determine the combined and individual effects of the ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth. We demonstrated that these two neurotrophic factors, when applied in a 1:1 combination, but not individually, induced directed growth of neuronal processes towards the source of the gradient. This chemotactic effect persists without significant changes over a wide (10-fold) concentration range. Moreover, we demonstrated that other, more general growth parameters that do not evaluate growth in a specific direction (such as, neurite length and trajectory) were differentially affected by the concentration of the CNTF/GNDF mixture. Furthermore, GDNF, when applied individually, did not have any chemotactic effect, but caused significant neurite elongation and an increase in the number of neurites per ganglion.

Assessing Axonal Degeneration in Embryonic Dorsal Root Ganglion Neurons In Vitro.

The molecular players regulating the axon degeneration pathway have been identified using in vitro experimental models. Here, we describe an in vitro assay to assess the axonal fragmentation induced by mechanical injury to axons in cultured mouse embryonic dorsal root ganglion (DRG) neurons. DRG neurons are pseudounipolar and therefore suitable for an assay of axonal degeneration after injury. In addition, the time course of the axonal fragmentation is stereotyped, enabling the identification of reagents that either expedite or impede the degeneration process. With an image-based quantification method, the in vitro degeneration assay can be utilized as a platform supporting high-throughput screens for pharmacological or genetic reagents delaying axon degeneration.

A Schwann Cell-Neuron Coculture System to Study Neuron-Glia Interaction During Axon Degeneration.

Autonomous mechanisms of axon degeneration are frequently studied in vitro by mechanical axon injury of isolated sensory neurons. This has led to major advances in understanding the molecular pathways governing axon degeneration. However, this approach does not pay attention to potential glial mechanisms for the regulation of axon death. Here, I describe a straightforward protocol to seed purified rat Schwann cells on neuronal cultures in order to study the interaction between axons and these glia during axon degeneration.

Long-lived zebrafish Rohon-Beard cells.

During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13-16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

Primary Cultures of Pure Embryonic Dorsal Root Ganglia Sensory Neurons as a New Cellular Model for Friedreich's Ataxia.

Peripheral neuropathies can have various origins, from genetic to acquired causes, and affect altogether a large group of people in the world. Current available therapies aim at helping the disease symptoms but not to correct or stop the development of the disease. Primary neuronal cultures represent an essential tool in the study of events related to peripheral neuropathies as they allow to isolate the affected cell types, often originating in complex tissues in which they account for only a few percentage of cells. They provide a powerful system to identifying or testing compounds with potential therapeutic effect in the treatment of those diseases. Friedreich's ataxia is an autosomal recessive neurodegenerative disorder, which is characterized by a progressive spinocerebellar and sensory ataxia. Proprioceptive neurons of the dorsal root ganglia (DRG) are the primary affected cells. The disease is triggered by a mutation in the gene FXN which leads to a reduction of the frataxin protein. In order to study the neurophysiopathology of the disease at the cellular and molecular levels, we have established a model of primary cultures of DRG sensory neurons in which we induce the loss of the frataxin protein. With such a model we can alleviate the issues related to the complexity of DRG tissues and low amount of sensory neuron material in adult mouse. Hereby, we provide a protocol of detailed and optimized methods to obtain high yield of healthy mouse DRG sensory neuron in culture.

Neuronally Enriched RUFY3 Is Required for Caspase-Mediated Axon Degeneration.

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.

Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx.

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro. We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca2+, and here, we address the mechanism of Ca2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca2+ influx in sensory axon degeneration.

Methods for fabrication and evaluation of a 3D microengineered model of myelinated peripheral nerve.

OBJECTIVE: The cost and low success rates of the neurological drug development pipeline have diverted the pharmaceutical industry to 'nerve-on-a-chip' systems as preclinical models to streamline drug development. We present a novel micro-engineered 3D hydrogel platform for the culture of myelinated embryonic peripheral neural tissue to serve as an effective in vitro model for electrophysiological and histological analysis that could be adopted for preclinical testing. APPROACH: Dorsal root ganglions (DRG) from 15 d old embryonic rats were cultured in 3D hydrogel platforms. The interaction between Schwann cells (SC) and neurons during axonal development and regeneration affects the direction of growth and the synthesis of myelin sheaths. Induction of myelination was performed with two approaches: the addition of exogenous SC and promoting migration of endogenous SC. MAIN RESULTS: Histological analysis of the preparation utilizing exogenous SC showed aligned, highly fasciculated axonal growth with noticeable myelin sheaths around axons. Separately, electrophysiological testing of the preparation utilizing endogenous SC showed increased amplitude of the compound action potential and nerve conduction velocity in the presence of ascorbic acid (AA). SIGNIFICANCE: This platform has immense potential to be a useful and translatable in vitro testing tool for drug discovery and myelination studies.

The dependence receptor TrkC regulates the number of sensory neurons during DRG development.

The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG.

Preparation of Neonatal Rat Schwann Cells and Embryonic Dorsal Root Ganglia Neurons for In Vitro Myelination Studies.

The ability to understand in great details, at the molecular level, the process of myelination in the peripheral nervous system (PNS) is, in no minor part, due to the availability of an in vitro culture model of PNS myelination. This culture system is based on the ability to prepare large population of highly purified Schwann cells and dorsal root ganglia neurons that, once co-cultured, can be driven to form in vitro well-defined myelinated axon units. In this chapter, we present our detailed protocols to establish these cell cultures that are derived from modifications of procedures developed 35-40 years ago.

Isolation and Expansion of Schwann Cells from Transgenic Mouse Models.

The most widely used method (Brockes' method) for preparing primary Schwann cell culture uses neonatal rat sciatic nerves as the primary source of Schwann cells. The procedure is relatively simple and yields a highly purified population of Schwann cells in a short period of time. The method has also been used to prepare Schwann cells from mice, however, with limitation. For example, Brockes' method is not applicable when the genotypes of mouse neonates are unknown or if the mouse mutants do not develop to term. We described a method ideal for preparing Schwann cells in a transgenic/knockout mouse study. The method uses embryonic dorsal root ganglia as the primary source of Schwann cells and allows preparing separate, highly purified Schwann cell cultures from individual mouse embryos in less than 2 weeks.

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system.

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target-derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret-expressing neurons in the DRG complete developmental cell death prior to TrkA-expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701-717, 2018.

Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat.

Cobalt is a trace element that localizes in the human body as cobalamin, also known as vitamin B12. Excessive cobalt exposure induces a peripheral neuropathy, the mechanisms of which are yet to be elucidated. We investigated how cobalt may affect mitochondrial motility in primary cultures of rat dorsal root ganglion (DRG). We observed mitochondrial motility by time-lapse imaging after DsRed2 tagging via lentivirus, mitochondrial structure using transmission electron microscopy (TEM), and axonal swelling using immunocytochemical staining. The concentration of cobaltous ion (Co2+) required to significantly suppress mitochondrial motility is lower than that required to induce axonal swelling following a 24-h treatment. Exposure to relatively low concentrations of Co2+ for 48 h suppressed mitochondrial motility without leading to axonal swelling. TEM images indicated that Co2+ induces mitochondrial destruction. Our results show that destruction of the axonal mitochondria precedes the axonal degeneration induced by Co2+ exposure.