The effects of galanin on dorsal root ganglion neurons with high glucose treatment in vitro

The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy. The extracellular high concentration of glucose can cause neuronal cellular damage. Galanin (Gal) not only plays a role in processing of sensory information but also participates in energy homeostasis and glucoregulation. However, the effects of Gal on dorsal root ganglion (DRG) neurons with high glucose are not clear. Using an in vitro model of high glucose-treated DRG neurons in culture, the effects of Gal on intracellular reactive oxygen species (ROS) expression, cell viability, apoptosis, expression of Gal and its receptors (GalR1 and GalR2) of DRG neurons were investigated. Neurons were dissociated from embryonic day 15 (E15) rat DRG and cultured for 48 h and then maintained in serum-free neurobasal medium containing high glucose (45 mmol/L) or normal glucose (25 mmol/L) for 24 h. Mannitol (20 mmol/L) was also used to create a high osmotic pressure mimicking the high glucose condition. The results showed that high glucose caused a rapid increasing of intracellular ROS, decreases of cell viability, and upregulation of Gal and its mRNA. Exogenous Gal (1 μmol/L) inhibited the above effects caused by high glucose. Interestingly, high glucose caused downregulation of GalR1 and its mRNA and administration of exogenous Gal could further decrease their expression, whereas expression of GalR2 and its mRNA was not affected at different experimental conditions. The results of the present study indicate for the first time that Gal and its receptor system are involved in high glucose-induced DRG neuronal injury. The contribution of exogenous Gal on neuroprotection appears to be quite significant. These results provide rationale and experimental evidence for development and further studies of Gal on therapeutic strategy for improving diabetic neuropathy.     

周围神经损伤后脊神经节感觉神经元胞体形态学的变化

目的 研究周围神经损伤后脊神经节感觉神经元胞体形态学的变化以探讨其主要死广性质。方法 切断并原位吻合大鼠右侧坐骨神经，左侧不作任何处理，作为对照；于术后不同时间取L4-L6脊神经节作光镜和电镜观察，观察脊神经节感觉神经元胞体形态的变化。结果 光镜下，损伤的脊神经节感觉神经元胞体染色质浓染；电镜下，细胞膜内陷，分割细胞内容物成凋亡小体；而对侧脊神经节感觉神经元胞体均一、无变化。结论 大鼠坐骨神经损伤后，脊神经节感觉神经元有死亡，其胞体的形态学变化符合细胞凋亡特征。     

大鼠背根神经节细胞的分离及特性探讨

目的探讨大鼠背根神经节（dorsal root ganglion，DRG）细胞的分离方法以及细胞形态和电生理特征。方法采用显微外科技术获取大鼠DRG体，用双酶法急性分离大鼠DRG获得DRG细胞，全细胞膜片钳技术记录动作电位和钠电流。结果本实验能得到完整圆形或椭圆长条形的大鼠DRG体。正常的单个DRG细胞呈圆形或椭圆形，大小不等，胞膜清晰，折光性好，隐约可见细胞核。在DRG细胞上记录的动作电位都具有从0期到4期，呈正立锐角三角形，静息电位小，动作电位时程短。DRG细胞的钠通道最大电流密度在-30mV左右，几乎能被，TTX完全抑制，具有可逆性恢复。结论本实验采用分离方法简单易行，DRG细胞容易获得和辨认，适合膜片钳技术要求，电生理特征明确可靠，值得推崇。     

Epibatidine, a nicotinic acetylcholine receptor agonist, inhibits the capsaicin response in dorsal root ganglion neurons

In patch-clamp recordings from small-medium diameter dorsal root ganglion neurons in culture, (+/-)-epibatidine (1 microM) was able to inhibit the capsaicin response (IC(50)=0.32 microM) in neurons where there was no detectable direct nicotinic response. Thus, (+/-)-epibatidine may inhibit the vanilloid receptor in a manner that is not dependent upon nicotinic current activation, representing another mechanism by which such ligands could modulate vanilloid receptor signaling.     

Taxol counteracts colchicine blockade of axonal transport in neurites of cultured dorsal root ganglion cells

Taxol does not affect axonal transport in cultured neurites. Preincubation of this drug inhibits the effects of colchicine on axonal transport and morphology in cultured neurites. A reduction of the free tubulin density by preincubated taxol might prevent the disruption of microtubules by colchicine.     

Neuronal Fc-gamma receptor I mediated excitatory effects of IgG immune complex on rat dorsal root ganglion neurons

Pain often accompanies antigen-specific immune-related disorders though little is known of the underlying neural mechanisms. A common feature among these disorders is the elevated level of antigen-specific immunoglobulin (Ig) G in the serum and the presence of IgG immune complex (IC) in the affected tissue. We hypothesize that IC may directly activate the Fc-gamma receptor type I (FcγRI) expressed in nociceptive dorsal root ganglion (DRG) neurons and increase neuronal excitability thus potentially contributing to pain. Immunofluorescent labeling indicated that FcγRI, but not FcγRIIB or FcγRIII, was expressed in a subpopulation of rat DRG neurons including those expressing nociceptive markers. Calcium imaging revealed that the IC, but neither of the antibody (IgG) or antigen alone, produced an increase in intracellular calcium. This effect was abolished by the removal of the IgG Fc portion in the IC or the application of an anti-FcγRI antibody, suggesting a key role of the FcγRI receptor. Removal of extracellular calcium or depletion of intracellular calcium stores prevented the IC-induced calcium response. In whole-cell current-clamp recordings, IC depolarized the resting membrane potential, decreased the rheobase, and increased the number of action potentials evoked by a depolarizing current at 2× rheobase. In about half of the responsive neurons, IC evoked action potential discharges. These results suggest that a subpopulation of nociceptive neurons expresses functional FcγRI and that the activation of this receptor by IC increases neuronal excitability.     

Opioid regulation of intracellular free calcium in cultured mouse dorsal root ganglion neurons

Opioid agonists induced an increase in the intracellular free calcium concentration ([Ca²⁺]_i) or an inhibition of K⁺ (25 mM)-stimulated increase in [Ca²⁺]_i in different subsets of mouse dorsal root ganglion (DRG) neurons. The total neuronal population was grouped into three classes according to somatic diameter and defined as small (<16 μm), intermediate (16–25 μm), or large (>25 μm) neurons. Substance P-like immunoreactivity was detected mainly in the small and intermediate neurons. The δ, κ, and μ opioid receptor agonists [D-Ser², Leu⁵]enkephalin-Thr (DSLET), U69593, and [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO) each induced a transient increase in [Ca²⁺]_i in a small fraction (<30%) of neurons. The increases in [Ca²⁺]_i were blocked by the opioid antagonist naloxone. The dihydropyridine-sensitive calcium channel blocker nifedipine also blocked the increase in [Ca²⁺]_i induced by 1 μM DSLET. The rank order of potency (percentage of cells responding to each opioid agonist) was DSLET > U69593 > DAMGO. The opioid-induced increase in [Ca²⁺]_i was observed mainly in large neurons, with a low incidence in small and intermediate neurons. Opioid agonists also caused inhibition of K⁺-stimulated increases in [Ca²⁺]_i, which were blocked by naloxone (1 μM). Inhibition of the K⁺-stimulated increase by 1 μM DSLET or U69593 was greater in small and intermediate neurons than in large neurons. © 1996 Wiley-Liss, Inc.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

嗅鞘细胞对培养脊髓神经元生长状态的影响

目的研究嗅鞘细胞(OECs)对体外培养脊髓背根神经节神经元生长状态的影响。方法取新生大鼠脊髓背根神经节细胞与嗅鞘细胞共培养,在显微镜下观察神经元生长发育情况,染色后进行细胞计数,并测定细胞活性。结果共培养组细胞密度明显高于对照组,神经元胞体大而饱满,突起较长,细胞活性较高。结论嗅鞘细胞可明显促进体外培养脊髓背根神经节神经元的生长,提高细胞活性。     

Three types of sodium channels in adult rat dorsal root ganglion neurons

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.     

Expression of the vanilloid receptor TRPV1 in rat dorsal root ganglion neurons supports different roles of the receptor in visceral and cutaneous afferents

A combination of tracing and multiple color immunofluorescence revealed that 69% of rat dorsal root ganglion (DRG) neurons innervating the urinary bladder expressed the vanilloid receptor TRPV1. In contrast, only 32% of DRG neurons innervating the skin of the L6 dermatome expressed TRPV1. However, a similar fraction of visceral (60-62%) and of cutaneous (59-60%) TRPV1-positive DRG neurons expressed the peptidergic markers substance P and calcitonin gene-related peptide, while the fraction of TRPV1-positive neurons that was labeled by the non-peptidergic marker Isolectin B4 was 58% for cutaneous and only 24% for visceral afferents. These results underscore differences of expression of functional markers in visceral and cutaneous afferents and support different mechanisms of activation of TRPV1 in viscera and in skin.     

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons

Intracellular magnesium concentration ([Mg²⁺]_i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg²⁺]_i was 0.48±0.08 mM (mean±SEM, n=23) at rest, and it increased 3-fold by depolarization with a 60-mM K⁺ solution. The [Mg²⁺]_i increase was observed in the absence of extracellular Mg²⁺, but the increase disappeared in the absence of extracellular Ca²⁺. 50 μM cadmium or 100 μM verapamil, a Ca²⁺ channel blocker, also diminished the rise of [Mg²⁺]_i. The additional measurement of an intracellular Ca²⁺ concentration ([Ca²⁺]_i) indicated that the [Mg²⁺]_i rise requires a threshold concentration of [Ca²⁺]_i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca²⁺-influx through voltage dependent Ca channels and this causes the release of Mg²⁺ from intracellular stores into the cytoplasm.     

Axonal dystrophy of dorsal root ganglion sensory neurons in a mouse model of Niemann-Pick disease type C

Niemann-Pick disease type C (NP-C) is a progressive and fatal neurological disorder characterized by intracellular accumulation of cholesterol and glycolipid. A Balb/c-npc1 mutant strain is a genetically authentic murine model of NP-C, and homozygous mice show progressive weight loss and tremor or ataxia until death at 12-14 weeks of age. Neuropathologically, this model is known to faithfully reproduce the cardinal histologic features of NP-C including neuronal storage, appearance of swollen axons (spheroids), and neuronal loss, although the cellular mechanisms of neural degeneration are largely unknown. To investigate the mode of neural degeneration of sensory neurons in NP-C, we studied the central processes of dorsal root ganglion (DRG) neurons at the level of the medullary dorsal column nuclei and the spinal dorsal horn with special attention to the ultrastructural changes of presynaptic axon terminals. The appearance of axonal spheroids in the dorsal column nuclei and the loss of axons in the spinal nerve roots were assessed quantitatively. We show that the gracile nuclei develop numerous axonal spheroids after only 3 weeks. At 6 and 9 weeks, dystrophic axons, which were separated from simple axonal spheroids by the ultrastructural presence of distinctive tubulo-vesicular elements, progressively increased in size and number. These neuropathological findings are identical to those of gracile axonal dystrophy (GAD) of the normal aging mouse. Presynaptic elements were exclusively involved in spheroid formation. The cuneate nuclei and the spinal dorsal horn revealed fewer axonal spheroids and only rare dystrophic changes. This was associated with a significant drop in the number of L4-5 dorsal root axons in NP-C mouse at 9 weeks of age compared with controls. These results support the existence of a length-dependent axonopathy in the central processes of DRG neurons and are consistent with the view that altered axonal transport, which is implicated in the pathogenesis of GAD in physiological aging, may be an underlying mechanism in neuronal degeneration in NP-C. Clinically, the premature development of GAD may be responsible for ataxia, one of the early manifestations of this disease.     

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.     

Neurofibrillary degeneration in cultured adult mouse neurons induced by maytansine

Summary The effects of maytansine, an antimitotic compound isolated from an African plant, were studied by light and electron microscopy in dissociated cell cultures of adult mouse dorsal root ganglia.Maytansine at 10–100 ng/ml concentration caused reversible, concentration-dependent, inhibition of microtubule assembly and induction of a large amount of 10 nm filaments in the cytoplasm of cultured neurons and Schwann cells.This work was supported by USPHS grants NS-10648, NS-15205 and RCDA award NS-00151     

Warm cells revealed by microfluorimetry of Ca in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca²⁺ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca²⁺]_i from 52.5±9.5 nM (mean±S.D., n=18) to 171.0±15.6 nM in cells between 15 and 25 μm in diameter. The depletion of extracellular Ca²⁺ diminished the Ca²⁺ elevation. The Na⁺-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Differential effects of hexachlorocyclohexane isomers on the GABA receptor-chloride channel complex in rat dorsal root ganglion neurons

The GABA_A receptor-chloride channel complex has recently been demonstrated by patch clamp experiments to be the target of cyclodiene insecticides. We have now examined the effects of four isomers of hexachlorocyclohexane (HCH), α-, β-, γ- and δ-HCH, on the GABA_A receptor-chloride channel complex of rat dorsal root ganglion neurons using patch clamp techniques. When co-applied with 10 μM GABA, 1 μM γ-HCH slightly enhanced and then suppressed the GABA-induced chloride current. The desensitization of the current was greatly accelerated by γ-HCH in a dose-dependent manner. The acceleration of desensitization and the suppression of sustained component of current by γ-HCH occurred at lower concentration ranges than those for the suppression of peak current. When 10 μM δ-HCH was co-applied with 10 μM GABA, current was greatly enhanced and then suppressed, and the level of enhancement was much higher than that of γ-HCH. α- and β-HCH had little or no effect on the GABA-induced chloride current. The differential actions of these isomers on GABA-activated chloride currents account for variable symptoms of poisoning in insects and mammals.     

The role of altered sodium currents in action potential abnormalities of cultured dorsal root ganglion neurons from trisomy 21 (down syndrome) human fetuses

Trisomy 21 (Down syndrome) results in abnormalities in electrical membrane properties of cultured human fetal dorsal root ganglion (DRG) neurons. Action potentials have faster rates of depolarization and repolarization, with decreased spike duration, compared to diploid neurons. In order to analyze the faster depolarization rate observed in trisomic neurons, we examined sodium currents of cultured human fetal DRG neurons from trisomy 21 and control subjects, using the whole-cell patch-clamp technique. The neurons were replated in culture to reduce dendritic spines. Two components of the sodium current were identified: (1) a fast, tetrodotoxin (TTX)-sensitive current; and (2) a slow, TTX-resistant component. The inactivation curves of both current types in trisomic neurons showed a shift of approximately 10 mV towards more depolarized potentials compared to control neurons. Thus, whereas essetially all of the fast sodium channels were inactivated at normal resting potentials in control neurons, approximately 10% of these channels were available for activation in trisomy 21 cells. Furthermore, the fast current showed accelerated activation kinetics in trisomic neurons. The slow sodium current of trisomic neurons showed slower deactivation kinetics than control cells. No differences were observed between trisomic and control neurons in the maximal conductance or current densities of either fast or slow current components. These data indicate that the greater rate of depolarization in trisomy 21 neurons at resting potentials is primarily due to activation of residual fast sodium channels that also have a faster time course of activation.     

Do neuropeptides in the dorsal horn change if the dorsal root ganglion cell death that normally accompanies peripheral nerve transection is prevented?

Peripheral nerve section causes the death of dorsal root ganglion cells and changes in neuroactive peptides in the dorsal horn of the spinal cord. The relationship between these 2 events has not been previously studied, however. One approach would be to prevent sensory cell death and then determine changes in peptide immunoreactivity. To do this, transected rat sciatic nerve stumps were placed in an impermeable silicone tube for one month. The tube was then removed and after 30 additional days the cells were counted. The data indicate that no cell death occurred. We conclude that the sensory cells are first saved due to some factor present in the tube, and then after 30 days, the cells become independent of the tube and its contents. In these same animals, all of the peptides we examined were significantly changed. Four of the peptides, calcitonin gene-related peptide (CGRP), substance P (SP), cholecystokinin octapeptide (CCK) and galanin (GAL) were significantly depleted in the medial L4-L5 superficial dorsal horn, and vasoactive intestinal polypeptide (VIP) was significantly increased. We conclude that there are major changes in spinal peptide systems following peripheral nerve transection even if there is no accompanying death of sensory neurons. Thus we suggest that dramatic central changes in peptide immunoreactivity following peripheral nerve transection are independent of the sensory cell death that usually occurs in response to this injury.     

Nitric oxide inhibits GABA-evoked current in dorsal root ganglion neuron via PKG-dependent pathway

gamma-Aminobutyric acid (GABA) is considered a major inhibitory neurotransmitter in the generation of presynaptic inhibition at central terminals of primary afferent fiber (PAF), while it has also been established that nitric oxide (NO) may sensitize the terminals of nocisponsive PAFs and enhance neuropeptide release, possibly via mechanisms involving the activation of a cyclic guanidine monophosphate (cGMP)-dependent PKG. The present work was undertaken to explore the modulatory effect of sodium nitroprusside (SNP), a donor of NO, on GABA-evoked current of isolated adult rat dorsal root ganglion (DRG) neurons and the intracellular mechanism involved, by means of whole-cell patch clamp recording. The results showed that 1 mM SNP reversibly inhibited the inward current evoked by 0.1 mM GABA (-1.05 +/- 0.17nA vs. -0.63 +/- 0.11nA, n = 22, p < 0.01 or 0.1 mM muscimol a specific GABA(A) receptor agonist (-1.70 +/- 0.39 nA vs. -1.01 +/- 0.24 nA, n = 6, p < 0.05), which could be cancelled by simultaneous application of 1 mM methylene blue, an inhibitor of PKG. After preapplication of SNP with increasing concentrations 0.03, 0.1, 0.3, 1, and 3 mM), SNP inhibited both 0.1 mM GABA-evoked current (IC(50) = 0.2423 mM, n = 5) and 0.1 mM muscimol-evoked current (IC(50) = 0.3255 mM, n = 3) in DRG neurons in a dose-dependent manner. Therefore, it was suggested that PKG-dependent pathway may be involved in the NO-induced inhibition of GABA(A) receptor-mediated inward current in rat DRG neurons, which may be involved in the presynaptic disinhibition of nociceptive information induced by NO under certain conditions.