Effects of ion channel activity on development of dorsal root ganglion neurons

Studies of mouse dorsal root ganglion neurons in vitro demonstrate that ion channel function and regulation can influence a wide range of developmental processes. The work suggests that much as exposure to different trophic factors, the pattern of impulse activity a neuron experiences can have significant structural and functional effects during development. Studies concerning effects of ion channel activity on growth cone motility, axon fasciculation, synaptic plasticity, myelination, and intracellular signaling pathways regulating gene expression are presented in the context of changes in endogenous firing patterns during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 158–170, 1998     

Radiation-induced apoptosis in dorsal root ganglion neurons

Ionizing radiation (IR) results in apoptosis in a number of actively proliferating or immature cell types. The effect of IR on rat dorsal root ganglion (DRG) neurons was examined in dissociated cell cultures. After exposure to IR, embryonic DRG neurons, established in cell culture for six days, underwent cell death in a manner that was dose-dependent, requiring a minimum of 8 to 16 Gy. Twenty-five per cent cell loss occurred in embryonic day 15 (E-15) neurons, grown in cell culture for 6 days (“immature”), and then treated with 24 Gy IR. In contrast, only 2% cell loss occurred in E-15 neurons maintained in culture for 21 days ("mature") and then treated with 24 Gy IR. Staining with a fluorescent DNA-binding dye demonstrated clumping of the nuclear chromatin typical of apoptosis. Initiation of the apoptosis occurred within 24 h after exposure to IR. Apoptosis was prevented by inhibition of protein synthesis with cycloheximide. Apoptosis induced by IR occurred more frequently in immature than in mature neurons. Immature DRG neurons have a lower concentration of intracellular calcium ([Ca2+]i) than mature neurons. Elevation of [Ca2+]i by exposure to a high extracellular potassium ion concentration (35 μM) depolarizes the cell membrane with a resultant influx of calcium ions. The activation of programmed cell death after nerve growth factor (NGF) withdrawal is inversely correlated with [Ca2+]i in immature DRG neurons. When treated with high extracellular potassium, these immature neurons were resistant to IR exposure in a manner similar to that observed in mature neurons. These data suggest that [Ca2+]i modulates the apoptotic response of neurons after exposure to IR in a similar manner to that proposed by the “Ca2+ setpoint hypothesis” for control of NGF withdrawal-induced apoptosis.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion

After injection of the L7 dorsal root ganglion with ³H-leucine, fast axoplasmic transport carries some 3–5 × more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Endothelial cell influence on dorsal root ganglion cell formation

Although endothelial cells are known to produce neutrotrophic factors, endothelial cell influence on growth and survival of ganglion cells has not been documented. For this reason, a long-term culture technique was modified to obtain dorsal root ganglion (DRG) cells. Cells, among them neurons, were released from clusters into the medium for more than four weeks. These cells were grown together with endothelial cells either (1) in close contact as contiguous co-culture, or (2) on porous inserts for non-contiguous co-culture, and, finally, (3) without endothelial cells for ganglion cell culture. Samples from the cultures were stained for the nuclear Ki-67-antigen to detect proliferating cells, and for neurofilaments (NF) to verify the presence of DRG cells with and without mitotic figures. The contiguous co-culture contained three times as many mitotic DRG cells as other culture set ups. Nerve growth factor had no mitotic effect on the different DRG cultures, although it supported the growth of endothelial cells. It is concluded that a subpopulation of DRG cells is easily harvested from long-term DRG cultures. These DRG cells undergo mitosis when in direct contact with endothelial cells.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion.

After injection of the L7 dorsal root ganglion with 3H-leucine, fast axoplasmic transport carries some 3--5 x more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

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.     

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.     

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development.

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.     

Properties of dorsal root unmedullated fibers on the two sides of the ganglion

As an aid in the interpretation of the physiological properties of unmedullated nerve fibers, particularly those having their cells of origin in the dorsal root ganglia, more precise information about their morphology has been acquired through employment of the electron microscope. The appearance of the fibers in the skin nerves is described, with special reference to the structure of their sheaths; and a notation is made about the bearing of the axon-sheath relationship on the biophysical mechanism of conduction (p. 714). There is no basic difference between the sheath systems of the d.r.C and the s.C fibers. Attention is called to a point of similarity between the sheaths of unmyelinated and myelinated axons (p. 715). An assessment was made of the likelihood of interaction between the fibers. In action potentials showing temporal dispersion at several distances, the elevations appeared in their calculated positions. A model of a group of Schwann sheaths was constructed from successive electron microscope sections, showing that the lengths of the sheath branches are short in comparison with the wave lengths of the action potentials. Supported by these and other considerations, the argument is strongly in favor of the conclusion that among d.r.C fibers, as in other fibers, there is no cross-excitation between the axons. A new analysis of the size distribution of the fibers in a sural nerve was made from electron microscope pictures; and from the measurements the action potential was constructed. The result confirmed the view, previously expressed, that the velocities of conduction in the fibers can be precisely accounted for by multiplying the diameters by a constant. In the dorsal roots, the striking change that takes place in the appearance of the fibers and their disposition in the Schwann sheaths can be seen in Fig. 11. The axons partake of the special properties of the peripheral branches, which necessitated the creation of the subdivision of d.r.C fibers. But, their diameters are much smaller. At a set of reduced conduction velocities the configuration of the compound action potential in the nerves is repeated in the roots, with the root velocities still conforming to the size-velocity rule derived from nerve axons.     

不同生长底物对培养背根神经节细胞的影响（简报）

发育期细胞和细胞外基质(extracellular matrix,ECM)之间的相互作用调节着细胞的功能,包括细胞的迁移、细胞骨架的构建、细胞的增值和分化。神经元“移居”体外后,失去了在体内所依托的组织学关系,必须黏附于一个固相表面才能生存,所以神经元只有在包被基质的培养器皿上才能存活,     

Lentiviral gene transfer into the dorsal root ganglion of adult rats

Background

Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K⁺ channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K_2P channels after peripheral axotomy in mammals.

Results

Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.

Conclusions

In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.     

Sodium channels in cultured chick dorsal root ganglion neurons

Isolated Na currents were studied in cultured chick sensory neurons using the patch clamp technique. On membrane depolarization, whole cell currents showed the typical transient and voltage-dependent time course as in nerve fibres. Na currents appeared at about-40 mV and reached maximum amplitude at around-10 mV. At low voltages (-30 to 0 mV), their turning-on was sigmoidal and inactivation developed exponentially. The ratio of inactivation time constants was found to be smaller than in squid axons and comparable to that of mammalian nodes of Ranvier. Peak conductance and steady-state inactivation were strongly voltage-dependent, with maximum slopes at-17 and-40 mV, respectively. The reversal potential was close to the Nernst equilibrium potential, indicating a high degree of ion-selectivity for the channel. Addition of 3M TTX, or replacement of Na by Choline in the external bath, abolished these currents. Internal pronase (1 mg/ml) and N-bromoacetamide (0.4 mM) made inactivation incomplete, with little effect on its rate of decay.Single Na channel currents were studied in outside-out membrane patches, at potentials between-50 and-20 mV. Their activation required large negative holding potentials (-90 mV). They were fully blocked by addition of TTX (3 M) to the external bath. At-40 mV their mean open time was about 2ms and the amplitude distribution could be fitted by a single Gaussian curve, indicating the presence of a homogeneous population of channels with a conductance of 11±2 pS. Probability of opening increased and latency to first opening decreased with increasing depolarization. Inactivation of the channel became faster with stronger depolarizations, as measured from the inactivation time course of sample averages. Internal pronase (0.1 mg/ml) produced effects on inactivation comparable to those on whole cell currents. Openings of the channel had a tendency to occur in bursts and showed little inactivation during pulses of 250 ms duration. The open lifetime of the channel at low potentials (-50,-40 mV) was only three times larger than in control patches, suggesting that Na channels in chick sensory neurons can close several times before entering an inactivating absorbing state.     

Calcium- and dynamin-independent endocytosis in dorsal root ganglion neurons

Synaptic vesicle endocytosis is believed to require calcium and the GTPase dynamin. We now report a form of rapid endocytosis (RE) in dorsal root ganglion (DRG) neurons that, unlike previously described forms of endocytosis, is independent of calcium and dynamin. The RE is tightly coupled to calcium-independent but voltage-dependent secretion (CIVDS). Using FM dye and capacitance measurements, we show that membrane depolarization induces RE in the absence of calcium. Inhibition of dynamin function does not affect RE. The magnitude of RE is proportional to that of preceding CIVDS and stimulation frequency. Inhibitors of protein kinase A (PKA) suppress RE induced by high-frequency depolarization, while PKA activators enhance RE induced by low-frequency depolarization. Biochemical experiments demonstrate that depolarization directly upregulates PKA activity in calcium-free medium. These results reveal a calcium- and dynamin-independent form of endocytosis, which is controlled by neuronal activity and PKA-dependent phosphorylation, in DRG neurons.     

缓激肽对背根节神经元钠通道电流的作用

目的：观察缓激肽(bradykinin,BK)对大鼠背根节神经元电压依赖性钠通道电流的作用。方法：采用全细胞膜片钳技术,记录钠通道电流。结果：缓激肽剂量依赖性(0．01～10μmol／L)增高小细胞背根节神经元诱发放电频率;缓激肽剂量依赖性(O．01～10μmol／L)增加小细胞背根节神经元的河豚毒素不敏感(TTX—resistant,TTX—R)钠电流,对TTX敏感(TTX—sensitive,TTX-S)钠电流无明显影响。结论：缓激肽引起炎性痛的机制可能与TTX-R钠通道电流有关。     

Absence of neurogenesis of adult rat dorsal root ganglion cells

Recently, an age-related increase in the number of dorsal root ganglion (DRG) cells was reported in adult rats. This suggests neurogenesis of adult primary afferent neurons, which would be an extremely important phenomenon if it occurred. Other evidence is not compatible with this idea, however, so the issue is not settled. The primary point of contention concerns the counts of DRG cells in relation to age. In our opinion, these disagreements arise, at least in part, because different counting methods give different results for the same material. Thus, any method for determining DRG cell numbers should be calibrated. We previously calibrated some of the common methods used to count DRG cells and found that an empirical method gave accurate cell counts. In the present study, we have used this method and asked whether an age-related increase in the number of lumbar DRG cells can be demonstrated in adult rats. Our data indicate that DRG cell numbers remain essentially constant from 3 to 22 months of age. Most ancillary evidence is consistent with the hypothesis that mammalian DRG cell numbers do not change during adult life. Thus, we feel that the evidence does not support the hypothesis that there is neurogenesis of adult rat primary afferent neurons.