Adenine and guanine nucleotide content of Triton-extracted cytoskeletal fractions of nonmuscle cells.

Determination of the adenine and guanine nucleotides in Triton X-100-extracted cytoskeletal fractions was utilized to estimate the actin and tubulin content of the assembled cytoskeletons in nonmuscle cells. Results with stable cell lines (i.e., rat pheochromocytoma PC12 and neuroblastoma NB41A3) and with primary cultures (i.e., human foreskin fibroblasts and chick embryonic dorsal root ganglion neurons) exhibited levels of cytoskeletal fraction ADP and GDP consistent with their assembly-induced nucleoside-5'-triphosphatase activities only previously analyzed in vitro. Likewise, estimates of actin and tubulin content fall in the range of values obtained by other experimental approaches. In contrast, analysis of whole cell nucleotides showed high [ATP]/[ADP] and [GTP]/[GDP] ratios, suggesting there is little, if any, contamination of the cytoskeletal nucleotide pool by other cellular nucleotides.     

鸡胚背根神经节培养法测定神经生长因子活性的条件优化

鸡胚背根神经节培养法是测定神经生长因子(NGF)和神经营养因子活性的重要方法。我们通过考察培养基、鸡血浆和鸡胚浸液的比例、背根神经节部位、培养时间等条件对NGF刺激神经节生长的影响,从而建立了一套检测NGF活性的优化条件。在含20％鸡血浆和15％鸡胚浸液的DMEM的培养基中,利用伊莎褐鸡胚腰部的背根神经节,在终浓度为30ng/ml的蛇毒NGF的刺激下,培养36h可以得到理想的实验结果。该条件重复性好、分辨率高、简单实用。     

Effects of soluble laminin on organelle transport and neurite growth in cultured mouse dorsal root ganglion neurons: difference between primary neurites and branches

Laminin, an extracellular matrix molecule, is known to promote neurite growth. In the present study, the effects of soluble laminin on organelle transport and their relation to neurite growth were investigated in cultured dissociated mouse dorsal root ganglion (DRG) neurons. Laminin added into the extracellular medium was deposited on the surface of DRG neurons. DRG neurons incubated with soluble laminin exhibited branched, long, and thin neurites. Time-lapse study demonstrated that many small-diameter branches were newly formed after the addition of laminin. Thus, the growths of large-diameter primary neuritis, arising from cell bodies and branches extended from growth cones of primary neuritis, were analyzed separately. Laminin decreased the growth rate of primary neurites but increased that of branches. In primary neurites, acute addition of laminin rapidly decreased organelle movement in the neurite shaft and growth cone, accompanied by slowing of the growth cone advance. Branching of primary neurites occurred in response to laminin in some growth cones. In these growth cones, organelles protruded into nascent branches. In branches, soluble laminin increased organelle movement in the growth cone and the distal portion of the shaft. These results suggest that laminin inhibits the elongation of primary neurites but promotes branching and elongation of branches, all of which seem to be closely related to organelle transport.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

大鼠脊神经节内交感神经支配的荧光组织化学与HRP示踪观察

本研究应用乙醛酸诱发儿茶酚胺（ＣＡ）荧光技术观察大鼠肾上腺素（ＮＡ）能神经在脊神经节内的分布;并应用ＨＲＰ顺、逆行追踪技术对脊神经节内ＮＡ能神经纤维的起源及其与脊神经节神经元的关系进行了探讨。荧光组织化学观察发现、有些神经节神经元胞体周围分布有带膨体的ＮＡ能神经末梢;有的紧密围绕脊神经节细胞——卫星细胞复合体。颈上交感神经节内注射霍乱毒素Ｂ亚单位结合ＨＲＰ（ＣＢ┐ＨＲＰ）,在同侧Ｃ３～６节段脊神经节内可见标记的点状纤维末梢紧邻于节细胞旁。Ｔ１１～Ｌ２节段脊神经节内注射ＨＲＰ后,在同侧椎旁交感链（Ｔ９～Ｌ１）内可见标记的交感节后神经元胞体。上述实验结果表明,交感节后神经元发出节后纤维可直接到达脊神经节内,与节细胞发生接触。本研究提示、交感神经在脊神经节水平可能参与躯体初级传入信息的调制     

Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method

The developmental morphology of the hypoglossal nerve and associated structures were studied in the chick embryo (Hamburger and Hamilton stages 16-27) stained by the immunohistochemical technique. Ventral rootlets of the occipital nerves, including O1, were seen at stage 16. The distal ends of these nerves anastomosed to form the hypoglossal nerve at stage 20. At stage 23, four occipital and the first three cervical nerves were observed to be involved. The transient contribution of C3 at this stage seemed to be correlated with the formation of the longitudinal anastomosis of the distal end of the spinal nerves which begins around stage 23. The anterior hypoglossal roots appeared between O1 and the abducens nerve at stage 20. These rootlets were observed to arise as the rostral continuation of the occipital sequence and were found to be arranged in a straight line from O1 to the abducens nerve. The recurrent branch of the abducens was also observed. The posterior end of the ganglion crest produced dorsal root ganglion (DRG)-like structures transiently at the level of C2, and sometimes at the level of C1 also. The ganglion crest developed descending processes in the occipital region seemingly related to the spinal dorsal root formation. These phenomena seemed to represent the potential of the ganglion crest to produce the spinal nerve components which are depressed in the occipital region.     

Detection of basal and potassium-evoked acetylcholine release from embryonic DRG explants

Spontaneous and potassium-induced acetylcholine release from embryonic (E12 and E18) chick dorsal root ganglia explants at 3 and 7 days in culture was investigated using a chemiluminescent procedure. A basal release ranging from 2.4 to 13.8 pm/ganglion/5 min was detected. Potassium application always induced a significant increase over the basal release. The acetylcholine levels measured in E12 explants were 6.3 and 38.4 pm/ganglion/5 min at 3 and 7 days in culture, respectively, while in E18 explant cultures they were 10.7 and 15.5 pm/ganglion/5 min. In experiments performed in the absence of extracellular Ca2+ ions, acetylcholine release, both basal and potassium-induced, was abolished and it was reduced by cholinergic antagonists. A morphometric analysis of explant fibre length suggested that acetylcholine release was directly correlated to neurite extension. Moreover, treatment of E12 dorsal root ganglion-dissociated cell cultures with carbachol as cholinergic receptor agonist was shown to induce a higher neurite outgrowth compared with untreated cultures. The concomitant treatment with carbachol and the antagonists at muscarinic receptors atropine and at nicotinic receptors mecamylamine counteracted the increase in fibre outgrowth. Although the present data have not established whether acetylcholine is released by neurones or glial cells, these observations provide the first evidence of a regulated release of acetylcholine in dorsal root ganglia.     

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.     

Morphological and functional properties of rat dorsal root ganglion cells cultured in a chemically defined medium

A culture procedure for dorsal root ganglion (DRG) cells is presented using a completely defined culture medium without antibiotics, in combination with mechanical dissociation procedures. This culture procedure allows all dorsal root ganglion cell types to be cocultured for periods of at least 106 days. Some of the dorsal root ganglion neurons, which could be identified by their neurofilaments and the presence of fluoride resistant acid phosphatase, regained their original T-cell appearance within two weeks. After one month in culture ganglion-like reaggregates appeared. Schwann cells, satellite cells and fibroblasts were identified using morphological criteria. All neurons tested maintained excitability during, at least, the first 35 days in culture, since in all cases action potentials could be evoked by current pulses. The method has proved to be useful in the study of morphological, cytochemical and electrophysiological aspects of dorsal root ganglion cell differentiation in vitro.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Localization of organelle DNA synthesis within the root apical meristem of rice

DNA synthesis in cell nuclei and organelles in the root apicalmeristem of rice was analysed by anti-BrdU immunofluorescencemicroscopy to determine whether there is a specific order ofthese events in monocot roots. In the root meristem, organelleDNAs were synthesized in a specific region in the distal partof the root apical meristem, and were not synthesized in theroot meristem‘s proximal region or the elongation zone.In contrast, cell nuclear DNA was synthesized throughout theroot apical meristem, except in the quiescent centre. In theroot cap of rice, DNA synthesis in both cell nuclei and organellenucleoids was detected only in the two layers of cells at theproximal end, which is a striking characteristic of monocotyledonousplants. Moreover, to determine quantitatively the activity ofDNA synthesis in cell nuclei and organelle nucleoids in micro-scalesections of plant tissues, we developed novel techniques formicro-scale hybridization and immuno-detection analysis. Atthe distal end of the root apical meristem, DNA levels of plastidsand mitochondria were 4-fold and 5-fold greater than those inthe elongation zone, respectively. Intracellular organelle DNAlevels dropped rapidly as the distance from the root tip increased.The activity of organelle DNA synthesis in the distal end ofthe root apical meristem was about 10-fold greater than thatin the elongation zone. Our present results confirm that nuclearand organelle DNA synthesis are not synchronized, but the latteroccurs preferentially before multiple cell divisions. Key words: Organelle DNA synthesis, organelle nucleoids (organelle nuclei), root apical meristem, anti-bromo-deoxyuridine immunofluorescence microscopy, rice.     

Delta/Notch signaling promotes formation of zebrafish neural crest by repressing Neurogenin 1 function

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion sensory neurons; however, Rohon-Beard neurons and dorsal root ganglion neurons are not necessarily derived from the same precursor cell. We propose that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons.     

The actions of serotonin on frog primary afferent terminals and cell bodies

The actions of serotonin (5-HT) were studied in the isolated frog spinal cord and dorsal root ganglion preparations. In the spinal cord, 5-HT increased the spontaneous activity recorded from dorsal roots, facilitated evoked spinal reflexes and produced fast and slow primary afferent depolarization (PAD). A direct action of 5-HT on primary afferent terminals is likely since 5-HT induced PAD remained in the presence of 1 microM tetrodotoxin and 2 mM Mn2+. The direct action of 5-HT on primary afferent terminals was blocked by methysergide and attenuated by concentrations of Mn2+ in excess of that required to block transmitter release. Cell bodies of the dorsal root ganglion were also depolarized by 5-HT. A slow hyperpolarization occasionally followed the initial depolarization. The depolarizing action of 5-HT in the dorsal root ganglion was also attenuated by treatment with Mn2+. It is concluded that 5-HT acts directly on frog primary afferents and that this influence may involve a calcium sensitive process. The dorsal root ganglion response to 5-HT appears to be a suitable model of the afferent terminal response.     

Quantitative study on the fetal cervical dorsal root ganglion and observations on its relationship with the spinal nerve complex in the albino mouse

In mouse fetuses aged 12, 14, 16, 18 and 20 days, the cervical dorsal root ganglion was studied quantitatively. The main growth in volume of the interneuroblastic spaces was between the 12th and 16th day of pre-natal life while the main increase in volume of its neuroblasts occurred in the subsequent 4 days. Thus, it was postulated that the growth and branching of the neuroblastic dendrites, growth of the neuroglial elements and the vascular ramifications inside the ganglion occurred mainly between the 12th and 16th day of pre-natal life. Different modalities in the spatial relationship between the dorsal root ganglion and the different components of the spinal nerve were met with. At times, the trunk of the spinal nerve was located inside the ganglion. At that site, the posterior primary ramus emerged from it and appeared as a branch of the ganglion. The ventral root sometimes passed close to the fibrous capsule of the ganglion. In other cases, it passed inside the ganglion, dividing the ganglionic neuroblasts into dorsal and ventral groups. These either remained ensheathed by one fibrous capsule or became divided into two separate masses that remained connected to each other by the fibrous dural sheath.     

Frataxin deficiency induces Schwann cell inflammation and death

Mutations in the frataxin gene cause dorsal root ganglion demyelination and neurodegeneration, which leads to Friedreich's ataxia. However the consequences of frataxin depletion have not been measured in dorsal root ganglia or Schwann cells. We knocked down frataxin in several neural cell lines, including two dorsal root ganglia neural lines, 2 neuronal lines, a human oligodendroglial line (HOG) and multiple Schwann cell lines and measured cell death and proliferation. Only Schwann cells demonstrated a significant decrease in viability. In addition to the death of Schwann cells, frataxin decreased proliferation in Schwann, oligodendroglia, and slightly in one neural cell line. Thus the most severe effects of frataxin deficiency were on Schwann cells, which enwrap dorsal root ganglia neurons. Microarray of frataxin-deficient Schwann cells demonstrated strong activations of inflammatory and cell death genes including interleukin-6 and Tumor Necrosis Factor which were confirmed at the mRNA and protein levels. Frataxin knockdown in Schwann cells also specifically induced inflammatory arachidonate metabolites. Anti-inflammatory and anti-apoptotic drugs significantly rescued frataxin-dependent Schwann cell toxicity. Thus, frataxin deficiency triggers inflammatory changes and death of Schwann cells that is inhibitable by inflammatory and anti-apoptotic drugs.     

Chloroquine Intoxication Induces Ganglioside Storage in Nervous Tissue: A Chemical and Histopathological Study of Brain, Spinal Cord, Dorsal Root Ganglia, and Retina in the Miniature Pig

Abstract :
The effect of chronic chloroquine intoxication on lipid composition, particularly the gangliosides, was studied in the nervous system of miniature pigs, type Göttingen. The tissues examined were cerebrum, spinal cord, dorsal root ganglia and retina. Chloroquine was given in the diet in doses of 2.0-3.5 g/kg food. The intoxication of the pigs was started at the age of 100–240 days and continued for 177-219 days. The control pigs received the same diet without chloroquine. The ganglioside concentration was increased in all the tissues examined. Dorsal root ganglia and retina were the tissues affected most and showed a twofold increase. This corresponded to the light and electron microscopically demonstrated extensive storage process in the perikarya of dorsal root ganglion cells and inner ganglion cells of the retina. Under light microscopy the storage material was granular, intensely PAS-positive and dissolved by paraffin embedding. The electron microscopical equivalent consisted of conglomerates of membranous lysosomal residual bodies. In cerebrum the ganglioside concentration was increased by 12%. Storage in the brain varied widely between different systems and types of cells. The allocortex was much more affected than the isocortex. Certain inhibitory ganglion cell types, such as the basket cells, exhibited the most massive storage of all. The spinal medulla was morphologically less involved but showed approximately the same ganglioside increase, though not statistically significant. With the exception of cerebrum the increase in the tissues examined involved all the individual gangliosides, most severely ganglioside GM2 and three fucogangliosides. In cerebrum only the ganglioside GM2 was increased more than the other gangliosides. Chloroquine intoxication did not affect the concentration of phospholipids or cholesterol in the cerebrum, spinal cord or dorsal root ganglia, but in retina the acidic phospholipids were significantly increased.     

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.