Dorsal Root Ganglion Cultures Research Articles

An aligned 3D neuronal-glial co-culture model for peripheral nerve studies

In this study we report on the development of a 3D in vitro peripheral nerve model using aligned electrospun polycaprolactone fibre scaffolds manufactured under tightly controlled and reproducible conditions with uniform diameters of 1 μm, 5 μm and 8 μm. Fibres were characterized by SEM for diameter, density and alignment properties and formed in to scaffolds for 3D in vitro culture. Three different approaches were adopted using: i) neuronal or primary Schwann cell cultures alone; ii) neuronal and primary Schwann cells in co-culture and iii) isolated dorsal root ganglion cultures, containing both neuronal and Schwann cells, with immunohistochemical and 3D confocal microscopy analysis. Neurite guidance was evident on all fibres diameters with the longest neurites detected on 8 μm fibres when cultured alone. However, co-culture with primary Schwann cells was found to enable neurite formation on all scaffolds. Dorsal root ganglion explants when grown on scaffolds showed both organised aligned neurite guidance and notably the co-localization of Schwann cells with neurites. Neurite lengths of up to 2.50 mm were routinely observed using 1 μm diameter fibres after 10 days and all cultures demonstrated a migrating Schwann cell ‘front’ of up to 2.70 mm (1 μm diameter fibres). Thus, a direct relationship was found between fibre diameter, neurite outgrowth and Schwann cell morphology. This work therefore supports the use of aligned electrospun PCL microfibres for the development of 3D peripheral nerve models in vitro. We envisage such models having future value in a number of areas including developmental biology, disease studies and the design of devices and scaffolds for peripheral nerve repair.

A DLK And JNK Dependent Axon Self- Destruction Program Promotes Wallerian Degeneration

A DLK And JNK Dependent Axon Self- Destruction Program Promotes Wallerian Degeneration

Transforming Growth Factor-β1 Regulates Cdk5 Activity in Primary Sensory Neurons

In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na(+)/Ca(2+) channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca(2+) influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.

Mo1873 Catecholaminergic Innervation Regulates Calcium Mobilization and CREB Activation in DRG Neuronal and Glial Cells

The transcription factor CREB (cAMP response element-binding) is implicated to function as a molecular switch underlying neural plasticity, and is activated in the primary sensory neurons in dorsal root ganglia (DRG) during experimental colitis. Activation of CREB is regulated by a list of kinases including Ca2+/calmodulin-dependent protein kinases (CaMK). The neuronal mediators in the sensory pathways contributing to CREB activation in the DRG during colitis are not identified. Recent studies have suggested a role of 5-hydroxytryptamine (5-HT) receptors in mediating sensory activity and visceral hypersensitivity. The AIMS of this study are 1) to examine serotonergic innervation of the DRG during colitis; 2) to examine the expression of metabotropic 5-HT receptor 5-HT4 and its association with phospho (p) -CREB expression in the DRG; and 3) to examine the effects of 5-HT on CREB activation and 5-HT-induced signaling pathways in the DRG. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 7 following induction of colitis. The DRG explants were incubated with 5-HT in the presence or absence of various inhibitors. Western blot and immunohistochemistry techniques were used for examination of protein expression. RESULTS: At 7 days of colitis, we have observed robust serotonergic innervation of the L1-L2 DRG where CREB was also activated. The serotonergic fibers contained small and regularly spaced varicosities. The expression level of 5-HT4 was significantly increased in these DRGs, and was co-localized with p-CREB. Application of 5-HT (10 μM) to DRG culture in the presence of Pargyline (1 mM) increased the expression level of p-CREB. 5-HT-elicited CREB activation was blocked by KN-62 (5 μM) and KN-93 (5 μM), the specific inhibitors of CaMKII. During colitis, the phosphorylation level of CaMKII was also increased in DRG neurons expressing p-CREB. CONCLUSION: TNBS colitis-induced CREB activation in primary sensory neurons is likely mediated by 5-HT-induced Ca2+/calmodulin-dependent pathways.

Mo1872 Enhanced Serotonergic Innervation of DRG During Colitis and Its Role in CaMKII-Mediated CREB Activation

The transcription factor CREB (cAMP response element-binding) is implicated to function as a molecular switch underlying neural plasticity, and is activated in the primary sensory neurons in dorsal root ganglia (DRG) during experimental colitis. Activation of CREB is regulated by a list of kinases including Ca2+/calmodulin-dependent protein kinases (CaMK). The neuronal mediators in the sensory pathways contributing to CREB activation in the DRG during colitis are not identified. Recent studies have suggested a role of 5-hydroxytryptamine (5-HT) receptors in mediating sensory activity and visceral hypersensitivity. The AIMS of this study are 1) to examine serotonergic innervation of the DRG during colitis; 2) to examine the expression of metabotropic 5-HT receptor 5-HT4 and its association with phospho (p) -CREB expression in the DRG; and 3) to examine the effects of 5-HT on CREB activation and 5-HT-induced signaling pathways in the DRG. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 7 following induction of colitis. The DRG explants were incubated with 5-HT in the presence or absence of various inhibitors. Western blot and immunohistochemistry techniques were used for examination of protein expression. RESULTS: At 7 days of colitis, we have observed robust serotonergic innervation of the L1-L2 DRG where CREB was also activated. The serotonergic fibers contained small and regularly spaced varicosities. The expression level of 5-HT4 was significantly increased in these DRGs, and was co-localized with p-CREB. Application of 5-HT (10 μM) to DRG culture in the presence of Pargyline (1 mM) increased the expression level of p-CREB. 5-HT-elicited CREB activation was blocked by KN-62 (5 μM) and KN-93 (5 μM), the specific inhibitors of CaMKII. During colitis, the phosphorylation level of CaMKII was also increased in DRG neurons expressing p-CREB. CONCLUSION: TNBS colitis-induced CREB activation in primary sensory neurons is likely mediated by 5-HT-induced Ca2+/calmodulin-dependent pathways.

Reduced translocation of the TRPv1 receptor in afferent neurons of cardiomyopathic rats

Heart failure (HF) patients exhibit an abnormal exercise pressor reflex (EPR) in response to exercise. Previous studies have implicated the TrpV1 receptor, a vanilloid receptor and transmembrane ion channel, as a mediator of the EPR. A necessary step in activation of TRPv1 is translocation to the membrane. To determine if the translocation of TRPv1 is affected in HF, we compared the subcellular localization of TRPv1 in primary cultures of dorsal root ganglia (DRG) from normal and cardiomyopathic rats. DRGs (L4–L6) of normal or cardiomyopathic rats were evaluated in the presence or absence of NGF. Cells were stained for TrpV1 and fluorescence intensities of membrane and cytoplasm were quantified. Normal NGF treated DRG cells showed an increased ratio of membrane to cytoplasmic TrpV1 staining as compared to normal untreated control. In HF, the ratio of membrane to cytoplasmic TrpV1 was similar regardless of treatment. These results suggest that HF DRGs are impaired in their ability to recruit TrpV1 to the membrane. Furthermore, this suggests that abnormal EPR response in HF patients may be due, in part, to decreased TrpV1 activity due to reduced TrpV1 membrane localization.Studies supported by: NIH 2R01HL070242‐07A2 (MGG).

Activation of transient receptor potential A1 by a non‐pungent capsaicin‐like compound, capsiate

Capsiate is produced by 'CH-19 Sweet' (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels. Using Ca(2+) imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons. We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC(50) for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid. Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.

The microenvironment in the Hirschsprung's disease gut supports myenteric plexus growth

The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects. Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-β-1 (TGFβ-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated. The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.

Acute Morphine Induces Matrix Metalloproteinase-9 Up-Regulation in Primary Sensory Neurons to Mask Opioid-Induced Analgesia in Mice

BackgroundDespite decades of intense research efforts, actions of acute opioids are not fully understood. Increasing evidence suggests that in addition to well-documented antinociceptive effects opioids also produce paradoxical hyperalgesic and excitatory effects on neurons. However, most studies focus on the pronociceptive actions of chronic opioid exposure. Matrix metalloproteinase 9 (MMP-9) plays an important role in neuroinflammation and neuropathic pain development. We examined MMP-9 expression and localization in dorsal root ganglia (DRGs) after acute morphine treatment and, furthermore, the role of MMP-9 in modulating acute morphine-induced analgesia and hyperalgesia in mice.ResultsSubcutaneous morphine induced a marked up-regulation of MMP-9 protein in DRGs but not spinal cords. Morphine also increased MMP-9 activity and mRNA expression in DRGs. MMP-9 up-regulation peaked at 2 h but returned to the baseline after 24 h. In DRG tissue sections, MMP-9 is expressed in small and medium-sized neurons that co-express mu opioid receptors (MOR). In DRG cultures, MOR agonists morphine, DAMGO, and remifentanil each increased MMP-9 expression in neurons, whereas the opioid receptor antagonist naloxone and the MOR-selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) suppressed morphine-induced MMP-9 expression. Notably, subcutaneous morphine-induced analgesia was enhanced and prolonged in Mmp9 knockout mice and also potentiated in wild-type mice receiving intrathecal injection of MMP-9 inhibitors. Consistently, intrathecal injection of specific siRNA targeting MMP-9 reduced MMP-9 expression in DRGs and enhanced and prolonged morphine analgesia. Subcutaneous morphine also produced heat hyperalgesia at 24 h, but this opioid-induced hyperalgesia was not enhanced after MMP-9 deletion or inhibition.ConclusionsTransient MMP-9 up-regulation in DRG neurons can mask opioid analgesia, without modulating opioid-induced hyperalgesia. Distinct molecular mechanisms (MMP-9 dependent and independent) control acute opioid-induced pronociceptive actions (anti-analgesia in the first several hours and hyperalgesia after 24 h). Targeting MMP-9 may improve acute opioid analgesia.

Electron Microscopy Analysis of Neurites Extending from Dorsal Root Ganglia in vitro following Exposure to Intervertebral Disc Cells

Nucleus pulposus cells from the intervertebral disc have been shown to have inhibiting effects on neurite outgrowth in vitro. The nucleus pulposus consists of at least 2 cell populations, notochordal cells and chondrocyte-like cells. The aim of this study was to analyze the morphology of the neurites, from rat dorsal root ganglia (DRG) in a culture system, after exposure of these 2 cell populations. DRG from perinatal rats was harvested and placed in culture dishes for 24 h. Nucleus pulposus cells from donor rats were separated into 2 populations and applied to the DRG and neurite culture for a further 24 h and compared to control cultures exposed to culture medium without cells. The DRG and neurites were thereafter prepared for scanning or transmission electron microscopy (SEM/TEM). Descriptive SEM and TEM analyses and calculations of the neurite diameter were performed. The visual appearance after SEM and TEM preparation was similar in the three different culture conditions. However, there was a statistically significant reduction of the neurite diameter for the cultures exposed to notochordal cells compared to the cultures exposed to medium and chondrocyte-like cells (TEM preparation). Prominent and frequent pathologic abnormalities in peripheral nerve diseases have been observed with changes in axonal caliber. This study may suggest that a preserved small amount of notochordal cells, as seen in human adults, may play a role in clinical situations where nerve tissue is exposed to disc material, i.e. in disc herniation or degeneration.

Dicer-microRNA pathway is critical for peripheral nerve regeneration and functional recovery in vivo and regenerative axonogenesis in vitro

Both central and peripheral axons contain pivotal microRNA (miRNA) proteins. While recent observations demonstrated that miRNA biosynthetic machinery responds to peripheral nerve lesion in an injury-regulated pattern, the physiological significance of this phenomenon remains to be elucidated. In the current paper we hypothesized that deletion of Dicer would disrupt production of Dicer-dependent miRNAs and would negatively impact regenerative axon growth. Taking advantage of tamoxifen-inducible CAG-CreERt:Dicerfl/fl knockout (Dicer KO), we investigated the results of Dicer deletion on sciatic nerve regeneration in vivo and regenerative axon growth in vitro. Here we show that the sciatic functional index, an indicator of functional recovery, was significantly lower in Dicer KO mice in comparison to wild-type animals. Restoration of mechanical sensitivity recorded in the von Frey test was also markedly impaired in Dicer mutants. Further, Dicer deletion impeded the recovery of nerve conduction velocity and amplitude of evoked compound action potentials in vitro. Histologically, both total number of regenerating nerve fibers and mean axonal area were notably smaller in the Dicer KO mice. In addition, Dicer-deficient neurons failed to regenerate axons in dissociated dorsal root ganglia (DRG) cultures. Taken together, our results demonstrate that knockout of Dicer clearly impedes regenerative axon growth as well as anatomical, physiological and functional recovery. Our data suggest that the intact Dicer-dependent miRNA pathway is critical for the successful peripheral nerve regeneration after injury.

Multi-walled carbon nanotubes inhibit regenerative axon growth of dorsal root ganglia neurons of mice

Recent observations have demonstrated that nanomaterials may be toxic to human tissue. While the ability of nano-scaled particulate matter is known to cause a range of problems in respiratory system, recent observations suggest that the nervous system may be vulnerable as well. In the current paper we asked whether exposure of primary neuronal cell cultures to nanoparticles might compromise regenerative axon growth. Regenerative response was triggered by performing a conditioning lesion of sciatic nerve five days prior to collection of dorsal root ganglia (DRG). DRG neurons were plated at a low density and incubated with multi-walled carbon nanotubes (MWCNTs) (0.1–10 μg/ml in 10% of surfactant in saline) overnight. The experiments showed that exposure of DRG cultures to MWCNT significantly impaired regenerative axonogenesis without concomitant cell death. These results indicate that MWNCTs may have detrimental effect on nerve regeneration and may potentially trigger axonal pathology.

Relationship of Axonal Voltage-gated Sodium Channel 1.8 (NaV1.8) mRNA Accumulation to Sciatic Nerve Injury-induced Painful Neuropathy in Rats

Painful peripheral neuropathy is a significant clinical problem; however, its pathological mechanism and effective treatments remain elusive. Increased peripheral expression of tetrodotoxin-resistant voltage-gated sodium channel 1.8 (NaV1.8) has been shown to associate with chronic pain symptoms in humans and experimental animals. Sciatic nerve entrapment (SNE) injury was used to develop neuropathic pain symptoms in rats, resulting in increased NaV1.8 mRNA in the injured nerve but not in dorsal root ganglia (DRG). To study the role of NaV1.8 mRNA in the pathogenesis of SNE-induced painful neuropathy, NaV1.8 shRNA vector was delivered by subcutaneous injection of cationized gelatin/plasmid DNA polyplex into the rat hindpaw and its subsequent retrograde transport via sciatic nerve to DRG. This in vivo NaV1.8 shRNA treatment reversibly and repeatedly attenuated the SNE-induced pain symptoms, an effect that became apparent following a distinct lag period of 3-4 days and lasted for 4-6 days before returning to pretreatment levels. Surprisingly, apparent knockdown of NaV1.8 mRNA occurred only in the injured nerve, not in the DRG, during the pain alleviation period. Levels of heteronuclear NaV1.8 RNA were unaffected by SNE or shRNA treatments, suggesting that transcription of the Scn10a gene encoding NaV1.8 was unchanged. Based on these data, we postulate that increased axonal mRNA transport results in accumulation of functional NaV1.8 protein in the injured nerve and the development of painful neuropathy symptoms. Thus, targeted delivery of agents that interfere with axonal NaV1.8 mRNA may represent effective neuropathic pain treatments.

Effects of a synthetic bioactive peptide on neurite growth and nerve growth factor release in chondroitin sulfate hydrogels

Previous work has revealed robust dorsal root ganglia neurite growth in hydrogels of chondroitin sulfate. In the current work, it was determined whether addition of a synthetic bioactive peptide could augment neurite growth in these matrices via enhanced binding and sequestering of growth factors. Fluorescence recovery after photobleaching studies revealed that addition of peptide slowed nerve growth factor diffusivity in chondroitin sulfate gels, but not in control gels of hyaluronic acid. Furthermore, cultures of chick dorsal root ganglia in gels of hyaluronic acid or chondroitin sulfate revealed enhanced growth in chondroitin sulfate gels only upon addition of peptide. Taken together, these results suggest a synergistic nerve growth factor-binding activity between this peptide and chondroitin sulfate.

Up-regulation of dorsal root ganglia BDNF and trkB receptor in inflammatory pain: an in vivo and in vitrostudy

BackgroundDuring inflammation, immune cells accumulate in damaged areas and release pro-inflammatory cytokines and neurotrophins. Brain-derived neurotrophic factor (BDNF) plays a neuromodulatory role in spinal cord dorsal horn via the post-synaptic tyrosine protein kinase B (trkB) receptor to facilitate pain transmission. However, the precise role of BDNF and trkB receptor in the primary sensory neurons of dorsal root ganglia (DRG) during inflammation remains to be clarified. The aim of this study was to investigate whether and how BDNF-trkB signaling in the DRG is involved in the process of inflammatory pain.MethodsWe used complete Freund's adjuvant- (CFA-) induced and tumor necrosis factor-α- (TNF-α-) induced inflammation in rat hindpaw as animal models of inflammatory pain. Quantification of protein and/or mRNA levels of pain mediators was performed in separate lumbar L3-L5 DRGs. The cellular mechanism of TNF-α-induced BDNF and/or trkB receptor expression was examined in primary DRG cultures collected from pooled L1-L6 DRGs. Calcitonin gene-related peptide (CGRP), BDNF and substance P release were also evaluated by enzyme immunoassay.ResultsCFA injection into rat hindpaw resulted in mechanical hyperalgesia and significant increases in levels of TNF-α in the inflamed tissues, along with enhancement of BDNF and trkB receptor as well as the pain mediators CGRP and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in DRG. Direct injection of TNF-α into rat hindpaw resulted in similar effects with retrograde transport of TNF-α along the saphenous nerve to DRG during CFA-induced inflammation. Primary DRG cultures chronically treated with TNF-α showed significant enhancement of mRNA and protein levels of BDNF and trkB receptor, BDNF release and trkB-induced phospho-ERK1/2 signal. Moreover, CGRP and substance P release were enhanced in DRG cultures after chronic TNF-α treatment or acute BDNF stimulation. In addition, we found that BDNF up-regulated trkB expression in DRG cultures.ConclusionsBased on our current experimental results, we conclude that inflammation and TNF-α up-regulate the BDNF-trkB system in DRG. This phenomenon suggests that up-regulation of BDNF in DRG may, in addition to its post-synaptic effect in spinal dorsal horn, act as an autocrine and/or paracrine signal to activate the pre-synaptic trkB receptor and regulate synaptic excitability in pain transmission, thereby contributing to the development of hyperalgesia.

Atomic Force Microscopy Study of the Arrangement and Mechanical Properties of Astrocytic Cytoskeleton in Growth Medium

Astrocytes are quite interesting to study because of their role in the development of various neurodegenerative disorders. The present work describes an examination of the arrangement and mechanical properties of cytoskeleton of living astrocytes using atomic force microscopy (AFM). The experiments were performed with an organotypic culture of dorsal root ganglia (DRG) obtained from a chicken embryo. The cells were cultivated on a gelatinous substrate and showed strong adhesion. AFM allows one to observe cytoskeleton fibers, which are interpreted as actin filaments and microtubules. This assumption is supported by confocal microscopy fluorescence imaging of α-tubulin and fibrillar actin. Mapping of the local Young's modulus of a living astrocyte showed that the stiff areas correspond to the sites where the cytoskeleton fibers are located. Thus, the data obtained indicate that AFM is a promising method to study neural cells cytoskeleton integrity and arrangement inin vitromodels of neurodegeneration.

Carbon Disulfide Inhibits Neurite Outgrowth and Neuronal Migration of Dorsal Root Ganglion In Vitro

ABSTRACTCarbon disulfide (CS2) is a neurotoxic industrial solvent and widely used in the vulcanization of rubber, rayon, cellophane, and adhesives. Although the neurotoxicity of CS2 has been recognized for over a century, the precise mechanism of neurotoxic action of CS2 remains unknown. In the present study, a embryonic rat dorsal root ganglia (DRG) explants culture model was established. Using the organotypic DRG cultures, the direct neurotoxic effects of CS2 on outgrowth of neurites and migration of neurons from DRG explants were investigated. The organotypic DRG cultures were exposed to different concentrations of CS2 (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L). The number of nerve fiber bundles extended from DRG explants decreased significantly in the presence of CS2 (0.01 mmol/L, 15.00 ± 2.61, p < .05; 0.1 mmol/L, 11.17 ± 1.47, p < .001; 1 mmol/L, 8.00 ± 1.41, p < .001) as compared with that in the absence of CS2 (17.83 ± 2.48). The number of neurons migrated from DRG explants decreased significantly in the presence of CS2 (0.01 mmol/L, 79.50 ± 9.40, p < .01; 0.1 mmol/L, 62.50 ± 14.15, p < .001; 1 mmol/L, 34.67 ± 7.58, p < .001) as compared with that in the absence of CS2 (99.33 ± 15.16). And also, the decreases in the number of nerve fiber bundles and migrated DRG neurons were in a dose-dependent manner of CS2. These data implicated that CS2 could inhibit neurite outgrowth and neuronal migration from DRG explants in vitro.

Image-based Screening Identifies Novel Roles for IκB Kinase and Glycogen Synthase Kinase 3 in Axonal Degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.

Differential gene expression of neonatal and adult DRG neurons correlates with the differential sensitization of TRPV1 responses to nerve growth factor

Cultures of neonatal and adult dorsal root ganglion (DRG) neurons are commonly used in in vitro models to study the ion channels and signaling events associated with peripheral sensation under various conditions. Differential responsiveness between neonatal and adult DRG neurons to physiological or pathological stimuli suggests potential differences in their gene expression profiles. We performed a microarray analysis of cultured adult and neonatal rat DRG neurons, which revealed distinct gene expression profiles especially of ion channels and signaling molecules at the genomic level. For example, Ca 2+-stimulated adenylyl cyclase (AC) isoforms AC3 and AC8, PKCδ and CaMKIIα, the voltage-gated sodium channel β1 and β4, and potassium channels K v1.1, K v3.2, K v4.1, K v9.1, K v9.3, K ir3.4, K ir7.1, K 2P1.1/TWIK-1 had significantly higher mRNA expression in adult rat DRG neurons, while Ca 2+-inhibited AC5 and AC6, sodium channel Na v1.3 α subunit, potassium channels K ir6.1, K 2P10.1/TREK-2, calcium channel Ca v2.2 α1 subunit, and its auxiliary subunits β1 and β3 were conversely down regulated in adult neurons. Importantly, higher adult neuron expression of ERK1/2, PI3K/P110α, but not of TRPV1 and TrkA, was found and confirmed by PCR and western blot. These latter findings are consistent with the key role of ERK and PI3K signaling in sensitization of TRPV1 by NGF and may explain our previously published observation that adult, but not neonatal, rat DRG neurons are sensitized by NGF.

Expression, distribution and glutamate uptake activity of high affinity-excitatory aminoacid transporters in in vitro cultures of embryonic rat dorsal root ganglia

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (GLAST [glutamate aspartate transporter], GLT1 [glutamate transporter 1], EAAC1 [excitatory aminoacid transporter 1]) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Here we describe, through immunoblotting, immunofluorescence assays and β-counter analysis of [(3)H] l-glutamate uptake, the expression, distribution and activity of the glutamate transporters in in vitro cultures of embryonic dorsal root ganglia sensory neurons, sensory neurons+satellite cells and satellite cells. In this work we demonstrated that glutamate transporters are expressed in all cultures with a peculiar pattern of distribution. Even if GLAST is strongly detected in satellite cells, it is slightly expressed also in sensory neurons. GLT1 immunostaining is very weak in DRG neurons, but it was evident in the satellite cells. Finally, EAAC1 is localized in the soma and in the neuritis of sensory neurons, while it is not detectable in satellite cells. Moreover, all the cell cultures showed a strong sodium-energy-dependent glutamate uptake activity and it is more marked in neurons alone or in co-culture with satellite cells compared to satellite cells alone. Finally, we show that the complete or partial pharmacological inhibition of glutamate transporters virtually completely or partially abolish glutamate uptake in all cell culture. These results, that demonstrate that functionally active glutamate transporters can be studied in dorsal root ganglia cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any changes occur in in vitro models of peripheral nervous system damage.