Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons

Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (>98%) small (diameter <25 microm) and medium (diameter, 25-40 microm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66. 0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n=5). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast, alpha-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n=5), and also increased the instantaneous velocities of particles transported bidirectionally. Application of beta-CGRP (100-1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P and alpha-CGRP, both of which can be derived from its own and adjacent sensory neurons.     

Cell membrane expansion and blockade of action potentials produced by 2-decenoic acid in cultured dorsal root ganglion neurons

2-Decenoic acid, a fatty acid having 10 carbon atoms, blocks the action potentials of cultured dorsal root ganglion (DRG) neurons and this effect of 2-decenoic acid is reversible. From the analysis of the video pictures from Nomarski optics, relative values of the diameter and the thickness of the neurons increased to 1.06 and 1.14, respectively, when 2.1 mM 2-decenoic acid was applied to the neurons. The relative value of cell surface area, which was calculated from the equation for a spheroid, increased to about 1.20. On the other hand, relative fluorescence intensity of the fluorescent probe F18 (5-(octadecylthiocarbamoylamino)fluorescein) labeled neurons decreased to 0.81, when 2.1 mM 2-decenoic acid was applied to the neurons. This indicates that the relative cell surface area increased to 1.23, a value similar to that calculated from the results of the measurement of cell size. The time course of blocking action potentials after treatment of the fatty acid was similar to that of the cell membrane expansion. These results show that the fatty acid perturbs the cell membrane and expands the cell surface area and this expansion might reduce the opening ability of the Na+-channels in the membrane.     

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons were investigated by an immuno-cytofluorometric method. The receptor density showed a drastic decrease around birth and a smaller change after birth.     

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。     

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.     

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.     

Direct visualization and characterization of stable microtubules from the neurites of cultured dorsal root ganglion cells

We tested the stability of microtubules (MTs) in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under observation with a video-enhanced differential interference contrast (DIC) microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be MTs by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be MTs by specific immunostaining with anti-tubulin antibody. A significant number of MTs in the established neurites of 6-day-old cultures remained longer than 10 min after membrane removal while MTs in the Schwann cell processes or in the distal regions of the growth cone-bearing neurites of 3-day-old cultures disappeared within 2 min. A population of very stable MTs persisting longer than 30 min was also found specifically in the 6-day-old cultures. Association with other structures or bundling seemed to stabilize the MTs to some degree. The most stable MTs, however, were not associated with some structure along the length but were mainly anchored at points, suggesting that specific point attachments may be another important mechanism operating in MT stabilization. The present method is thus capable of directly demonstrating the unusual stability of neuritic MTs, and provides a new system for further investigation on the mechanism of stabilization. J. Neurosci. Res. 50:81–93, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Zinc (Zn) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1–5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at −80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn²⁺) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5–15 min, depending upin the concentration used. The IC₅₀ for inhibition of the peak current evoked by a step depolarization from −80 mV to 0 mV (N plus L channels) for 80 ms was 69 μM Zn²⁺ and the Hill slope about 1. The calcium current evoked by a voltage step from −80 mV to voltages between −40 mV and −15 mV (T-type current) was more sensitive (> 80% block with 20 μM Zn²⁺). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn²⁺ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.     

Spontaneous hyperpolarizations at the membrane of cultured mouse dorsal root ganglion cells

Intracellular recordings from cultured mouse dorsal root (sensory) ganglion cells revealed the presence of spontaneous hyperpolarizing potentials in over half of the cells. The potentials were resistant to tetrodotoxin and under voltage clamp were replaced by fluctuations in outward current. The power spectral density plots of these current fluctuations were predominantly of the Lorentzian type. These events may play an important role in the functioning of some sensory nerve cells.     

Selective axonal transport of anthracycline antibiotics

The retrograde intraaxonal transport of several anthracycline antibiotics was assessed in the peripheral nervous system of adult mice. Despite certain chemical similarities, the intraaxonal transport of these molecules was variable and could be correlated with the structure of the aminosugar at the R-2 position. One particularly interesting compound, 4′-deoxydoxorubicin, was transported by dorsal root ganglia axons, but not by the axons of ventral horn neurons. These observations suggest that the sugar component of a molecule may be important for its intraaxonal transport in the peripheral nervous system.     

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     

Spontaneous voltage and current fluctuations in tissue cultured mouse dorsal root ganglion cells

Fetal mouse dorsal root ganglion (DRG) neurons were maintained in primary dissociated cell culture for periods of 7 days to 3 months. Intracellular recordings from these cells revealed the presence of spontaneous subthreshold potentials in 101/177 neurons studied. When measured at the resting membrane potential, these spontaneous voltage events took two forms: (a) high frequency potential fluctuations several millivolts in peak-to-peak amplitude and (b) small, discrete hyperpolarizations. Neurons exhibiting either type of event were designated as 'active' DRG cells. No spontaneous potentials were seen in DRG cells hyperpolarized to membrane voltages more negative than -64 +/- 11.5 mV (n = 5 cells). Under voltage-clamp conditions, the subthreshold potentials of active DRG cells were replaced by fluctuations in outward current. The power spectral density, S(f) of these current fluctuations was approximated by an equation of the form S(f) = (S(o)/[1 + (f/fc) alpha] where 2 less than or equal to a less than or equal to 3 and the half-power frequency fc = 11.3 +/- 3.1 Hz at 23 degrees C (n = 17 cells). The spontaneous voltage fluctuations of active DRG cells were abolished in Ca2+-free saline, and of the divalent metal cations Sr2+, Mg2+, Ba2+, Co2+ and Mn2+, only Sr2+ could substitute for Ca2+ in the maintenance of this activity. Tetraethylammonium ions (1-10 mM) reversibly blocked the spontaneous potentials, while caffeine (10 mM) increased the frequency of these events. The spontaneous voltage fluctuations were not dependent on the presence of spinal cord neurons in the culture plate, and they were also observed in cultured DRG cells derived from adult mice.     

Freeze fracture analysis of the axolemma of cultured dorsal root ganglion neurons in the absence of Schwann cells

The distribution of intramembranous particles within the axolemma of cultured dorsal root ganglion neurons was determined by freeze-fracture microscopy. Utilizing culture conditions which eliminate Schwann cells, the particle distribution of the P-face, 735 +/- 119 microns2, and E-face, 100 +/- 39 microns2 resembled that of pre- and non-myelinated axons in vivo and no node-like E-face particle patching was seen. These results indicate that cultured neurite development is similar to that seen in vivo and that axons maintained in a glial-free environment do not develop nodal, E-face membrane specializations.     

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.     

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.     

A stereological study of dorsal root ganglion cells and nerve root fibers from rats treated with inorganic mercury

Unbiased stereological methods have been used to quantify the effects of inorganic mercury on the morphology of the fifth lumbar dorsal root ganglion cells and nerve root fibers. Adult male Wistar rats were treated with intraperitoneal injections of mercuric chloride (0.15 mg daily) for 30 days. The total numbers and mean volumes of A- and B-cell perikarya were estimated using the optical fractionator and the vertical rotator techniques. The total numbers of myelinated axons in the ventral and the dorsal roots were estimated with the two-dimensional fractionator technique and the areas of axon and myelin were estimated using the two-dimensional nucleator technique. No differences were found for any parameters in experimental and control animals, indicating that inorganic mercury intoxication alters neither the numbers or sizes of dorsal root ganglion cells and nerve root fibers nor the amount of myelin associated with the nerve fibers. Received: 20 January 1997 / Revised, accepted: 14 March 1997     

A stereological study of dorsal root ganglion cells and nerve root fibers from rats exposed to mercury vapor

Although mercury vapor is known to produce tremor and peripheral neuropathy, neuropathological studies of the effects of the vapor are few in number. The aim of the present study has been to evaluate the effect of mercury vapor on the morphology of the dorsal root ganglion and the spinal nerve roots. Adult male rats were exposed to mercury vapor for 33 days. The exposed rats developed somatic signs of intoxication and became increasingly irritable. The total numbers and volumes of A- and B-cell perikarya in the dorsal root ganglia, the total number of myelinated axons in the roots, and the cross-sectional areas of axon and myelin in the nerve roots were estimated using unbiased stereological principles. The mean cross-sectional area of myelin associated with nerve fibers in dorsal nerve roots of the exposed group was significantly reduced by 20% (2P = 0.014). A tendency towards a reduction was seen in axon area of myelinated nerve fibers in the dorsal nerve roots (2P = 0.087) and in the total numbers and mean volume of A-cell perikarya (2P = 0.059 and 2P = 0.087, respectively). No differences between the two test groups were found for any of the parameters measured in B-cells and ventral nerve roots. It is concluded that mercury vapor, in a dose sufficient to produce intoxication, induces only minor changes in dorsal root ganglion and nerve roots in rats. Received: 16 January 1998 / Revised, accepted: 23 Febraury 1998     

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.     

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。