MicroRNA-143 expression in dorsal root ganglion neurons

The unpleasant sensory and emotional experience of pain is initiated by excitation of primary afferent nociceptive neurons. Nerve damage or inflammation induces changes in nociceptive DRG neurons which contribute to both peripheral and central sensitization of pain-sensitive pathways. Recently, blockade of microRNA synthesis has been found to modulate the response of nociceptive neurons to inflammatory stimuli. However, little is known about the contributions of individual miRNAs to painful conditions. We compared miRNA expression in mouse sensory neurons and focussed on the localisation and control of miR-143. Using miRNA-arrays we compared the microRNA expression profile of intact lumbar DRG with one-day-old DRG cultures and found that nine miRNAs including miR-143 showed lower expression levels in cultures. Subsequent RT-qPCR confirmed array data and in-situ hybridisation localised miR-143 in the cytosol of sensory DRG neurons in situ and in vitro. Analysis of microbead-enriched neuron cultures showed significantly higher expression levels of miR-143 in isolectin B4 (I-B4) binding sensory neurons compared with neurons in the I-B4 negative flow-through fraction. In animal models of peripheral inflammation (injection of Complete Freund's Adjuvant, CFA) and nerve damage (transection of the sciatic nerve), we found that expression levels of miR-143 were significantly lower in DRGs ipsilateral to CFA injection or after nerve damage. Taken together, our data demonstrate for the first time miR-143 expression in nociceptive neurons. Since expression levels of miR-143 were higher in I-B4 positive neurons and declined in response to inflammation but not axotomy, miR-143 could selectively contribute to mRNA regulation in specific populations of nociceptors.     

A simulation study on the Ca2+-independent but voltage-dependent exocytosis and endocytosis in dorsal root ganglion neurons

In patch-clamped somata of dorsal root ganglion (DRG) neurons, two types of secretion have been proposed: Ca²⁺-dependent secretion and Ca²⁺-independent but voltage-dependent secretion (CIVDS). The Ca²⁺-induced and the depolarization-induced membrane capacitance (C_m) increases contribute 80 and 20% to the total C_m increase, respectively (Zhang and Zhou in Nat Neurosci 5:425, 2002). In order to explore the mechanism of the voltage-dependent C_m change (C_m), we constructed a model with sequential states. The simulation with this model closely approximates all the experimental data. The model predicts that the majority of fusion events (approximately 80%) are so-called kiss-and-run events, which account for the fast recovery or the rapid retrieval feature of the signals. The remaining 20% are attributed to full fusion events, which account for a slow retrieval feature. On the basis of the model, one mechanism of the activity-dependent endocytosis has revealed a differential distribution of vesicles between the kiss-and-run and full fusion states at different stimulation frequencies. The quantitative model presented in this study may help us to understand the mechanism of the CIVDS and the tightly coupled endocytosis found in mammalian DRG neurons.     

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.     

Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons

Two tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels, SNS and NaN, are preferentially expressed in small dorsal root ganglia (DRG) and trigeminal ganglia neurons, most of which are nociceptive, of rat and mouse. We report here the sequence of NaN from human DRG, and demonstrate the presence of two TTX-R currents in human DRG neurons. One current has physiological properties similar to those reported for SNS, while the other displays hyperpolarized voltage-dependence and persistent kinetics; a similar TTX-R current was recently identified in DRG neurons of sns-null mouse. Thus SNS and NaN channels appear to produce different currents in human DRG neurons.     

Absorption and retention characteristics of selenium in dorsal root ganglion neurons

Selenium concentration in the brain tissue is far less variable than those in peripherals, such as the liver and kidneys, in rodents, when fed a selenium-deficient diet. This fact implies the importance of this element for maintaining the integrity of brain functions and the distinctive selenium metabolism and/or the regulatory mechanism in the brain. To obtain basic information concerning the homeostatically maintained selenium store in the brain, we investigated absorption and retention characteristics of selenium from selenious acid (SA) and seleno-l-methionine (SeMet) in rat dorsal root ganglion (DRG) neurons, in comparison to isolated rat hepatocytes and renal cells in vitro. When DRG neurons were cultured in an SA-free medium subsequent to an SA-supplemented one for 24 h, the DRG neurons maintained a higher selenium concentration than that before SA supplementation over a period of 96 h after removal of SA from the culture medium. The cellular glutathione peroxidase activity of the cells increased for 72 h after removal of SA from the culture medium. A similar retention characteristic of selenium was also observed for DRG neurons treated with SeMet-supplemented culture medium. Consequently, selenium from source compounds, in part, was thought to be retained in DRG neurons and then be utilized for the synthesis of selenium-containing proteins, which implied the presence of a neuron-specific selenium retention mechanism.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40–48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40–48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

GPR35 is a functional receptor in rat dorsal root ganglion neurons

GPR35, previously an orphan G-protein coupled receptor, is a receptor for kynurenic acid. Here we examine the distribution of GPR35 in the rat dorsal root ganglion (DRG) and the effects of its selective activation. GPR35 was expressed predominantly by small- to medium-diameter neurons of the DRG. Many of these same neurons also expressed the transient receptor potential vanilloid 1 channel, a nociceptive neuronal marker. The GPR35 agonists kynurenic acid and zaprinast inhibited forskolin-stimulated cAMP production by cultured rat DRG neurons. Inhibition required G_i/o proteins as the effect was completely abolished by pretreatment with pertussis toxin. This is the first study to report the expression and function of GPR35 in rat nociceptive DRG neurons. We propose that GPR35 modulates nociception and that continued study of this receptor will provide additional insight into the role of kynurenic acid in pain perception.     

Membrane elasticity of mouse dorsal root ganglion neurons decreases with aging

Elastic area compressibility modulus (EACM) method was applied to measure the membrane elasticity of cultured dorsal root ganglion neurons from fetal and 3-month-old mice. The values of the EACM were 21.3 dyn/cm in 3-month-old neurons and 2.8 dyn/cm in fetal neurons. These results indicate that neural cell membrane elasticity decreases with aging.     

藜芦碱致使大鼠背根神经节A类神经元产生触发性振荡

在大鼠L5背根节浸浴钠通道失活门阻断剂藜芦碱（veratridine),记录该背根节神经元A类单纤维传入放电。发现：浸浴藜芦碱（1．8－3μmol/L）10min后,触压皮肤感受野或刺激坐骨神经引起部分静息纤维产生高频放电,其放电峰峰间期（interspike interval,ISI)形成U字形等型式的振荡,称之为触发性振荡。刺激脉冲的间隔越大,触发该振荡所需要的刺激脉冲数也就越多;不同时程和形式的刺激引起触发性振荡的形式无明显差异;触发性振荡的后抑制时期一般为30－90s。另外,实验还观察到该触发性振荡可由同一神经刺激引起的传入冲动触发。上述结果表明,用黎芦碱处理可使初级感觉神经元产生一种触发性振荡,该振荡机制可能与触发病的发作有关。     

Okadaic acid reversibly inhibits neurite outgrowth in embryonic dorsal root ganglion neurons

The aim of this study was to assess the effects of low concentrations of okadaic acid (OA) on neurite outgrowth and cellular integrity in cultures of dissociated dorsal root ganglion (DRG) neurons. The complete and fully reversible arrest of neurite outgrowth was achieved at 1 nM OA, thus ruling out the involvement of protein phosphatase 1 in the observed inhibitory effect. OA at 0.5 nM did not completely block neurite outgrowth, although it reduced the rate of growth by about one third. Protein phosphorylation and the integrity of microtubules and neurofilaments in neuron-enriched cultures were unaffected by 1 nM OA. The rate of synthesis of the low-molecular-weight neurofilament subunit (NFL) was also unchanged by OA treatment. Antimitotic agents used to eliminate proliferating cells did not alter the rate of neurite elongation. Since 1 nM OA does not suffice to inhibit neuronal protein phosphatase 2A fully, owing to the high concentration of this enzyme in neurons, we propose that the inhibitor is affecting a neuronal compartment that contains low levels of the phosphatase. This putative compartment is likely to be located in neurites, which were shown to contain levels of protein phosphatase 2A that were two- to threefold lower than in neuronal perikarya. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 193–201, 1997.     

Painful pathways induced by TLR stimulation of dorsal root ganglion neurons

We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1α, IL-1β, and PGE(2), which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain.     

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.     

Graphic representation of the distribution of acetylcholinesterase in cat dorsal root ganglion neurons

Synopsis Acetylcholinesterase (AChE) activity of primary sensory neurons of the cat has been quantitated and correlated with cell size. Dorsal root ganglia of the fourth and fifth thoracic spinal levels were studied. Frozen longitudinal and cross-sections were collected serially and stained with Cresyl Violet for total cell counts of the ganglia on the left; the average count was 3375 cells. Ganglia from the right were stained for AChE after the method of Karnovsky & Roots (1964) as modified by El Badawi & Schenk (1967), and counterstained with Haematoxylin. Cells were counted in every fourth section and the diameter of each was recorded. AChE-positive cells were classified as brown (B₁, B₂, B₃) and AChE-negative ones as blue (BL).An inverse correlation exists between cell size and AChE activity. High activity was demonstrated in 29% of the cells (B₁), moderate activity in 52% (B₂), minimal activity in 15% (B₃) and 4% were classified as AChE-negative (BL). Small cells with high activity were centrally located in the ganglia whereas large AChE-negative cells were peripherally distributed. Chi-Square analysis revealed that the size of the cell was not independent of the enzyme colour category.     

17beta-estradiol attenuates alpha, beta-meATP-induced currents in rat dorsal root ganglion neurons

The effects of 17beta-estradiol on the alpha,beta-me ATP-induced currents were studied on dorsal root ganglion (DRG) neurons using whole-cell recording technique. Three types of currents (transient, sustained or biphasic) were evoked by alpha,beta-me ATP in acutely dissociated DRG neurons. When neurons were pre-incubated with 17beta-estradiol (10-1000 nM) for 4 min, an inhibition of the transient current and the transient component of the biphasic current was observed. In contrast, 17beta-estradiol did not have any significant effect on the sustained current evoked by alpha,beta-meATP. The inhibitory effects were concentration-dependent, reversible and could be blocked by the estradiol receptor inhibitor, ICI 182,780 (1 microM). However, bovine serum albumin-conjugated 17beta-estradiol (17beta-estradiol-BSA, 10 nM) failed to mimic the effects of 17beta-estradiol. 17alpha-estradiol, the inactive isoform, did not have significant effects on alphabeta-meATP-induced currents, either. Sustained currents induced by ATP (100 microM) in nodose ganglion (NG), superior cervical ganglion (SCG) and otic ganglion (OTG) neurons were not affected by 17beta-estradiol. These results suggest that the female gonadal hormone, 17beta-estradiol, might participate in control of pain by modulating P2X3 receptor-mediated events in sensory neurons.