Avian parasympathetic neurotrophic factors: Age-related increases and lack of regional specificity

Parasympathetic neurons from 8-day-old chick embryo ciliary ganglia were grown in culture for 24 hr in the presence of extracts of chick heart from animals aged 7 days in ovo to 8 days posthatching. The biological activity of these cardiac extracts with respect to both neuronal survival and nerve fiber production increased with age of the donor animal to reach a plateau around hatching. Following polyacrylamide gel isoelectric focusing of posthatching chick heart and eye, a peak in activity that supports parasympathetic neuronal survival was found associated with eluates of gel slices of pH between 4.5 and 5.5 for both tissues. Our results suggest that the factor responsible for parasympathetic neuronal survival is common to at least two parasympathetic target organs.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

脊神经节感觉神经35kD特异蛋白的纯化

为纯化和鉴定感觉神经特异蛋白,以兔脊髓背根神经节及背根纤维组织为材料,通过制备匀浆、离子交换层析 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｃｅｌ,高压液相凝胶过滤层析分离纯化了脊神经感觉神经元 ３５ ｋ Ｄ蛋白,将其作为抗原制备抗 ３５ ｋ Ｄ 多克隆抗体． Ｗ ｅｓｔｅｒｎ ｂｌｏｔ 的结果表明,该蛋白特异地存在于脊感觉神经而不存在于脊运动神经．并初步观察到它对鸡胚背根节有神经营养作用．     

Neurite growth promoting factors of embryonic chick—ontogeny,regional distribution,and characteristics

A single neuronal cell biological assay for NGF has been used to quantitate factors in soluble extract of chick embryo that promote neurite outgrowth. The neurite growth promoting activity is detected as early as day 1.5 of embryonic life. Specific activity increases to maximum over the first four days and then decreases from day 5 to 6 during a growth spurt of the embryo. The tissue distribution of neurite growth promoting activity at eight embryonic days shows higher specific activity in peripheral structures that are richly innervated. Extract from all embryonic stages and from all tissues studied induces neurite outgrowth that is inhibited only partially by antibody to mouse NGF. Furthermore, the kinetics of neurite outgrowth and the neuronal populations responsive to extract differ from that of NGF. These observations indicate heterogeneity of the neurite growth promoting factors in soluble extracts of embryonic chick and demonstrate the presence of these factors before the responsive neurons leave the cell cycle and begin their differentiation processes.     

Heme oxygenase-2 in primary afferent neurons of the guinea-pig

Sensory ganglia (trigeminal, jugular, nodose, cervical and lumbar dorsal root ganglia) of the guineapig were investigated for the presence of a constitutive carbon monoxide-generating enzyme, heme oxygenase-2 (HO-2). A 36-kDa HO-2-immunoreactive protein was identified by Western blotting in protein extracts from dorsal root ganglia and localized by immunohistochemistry to all neuronal perikarya, including both substance P-positive and substance P-negative neurons, in all ganglia investigated. This ubiquitous distribution points to a general requirement for HO-2 in primary afferent neurons rather than to an association with a specific functionally defined subpopulation. Neither the axons of the sensory neurons nor their peripheral terminals in the skin and around visceral arteries were HO-2 immunoreactive. Explants of dorsal root ganglia with crushes placed on the dorsal roots showed accumulation of the neuropeptide, substance P, at the ganglionic side of the crush, but these axons were non-reactive to HO-2, indicating that there is no substantial transport of HO-2 towards the central ending of these sensory neurons. Collectively, the findings suggest that HO-2 exerts it major effects within the sensory ganglia themselves.     

具有NGF活性通过二硫键连接的 2个小分子多肽的制备及鉴定(英文)

神经生长因子 (NGF)作用广泛 ,β NGF是神经生长因子的活性部分 .由于 3对二硫键的影响 ,体外表达NGF很难形成正确折叠的肽链 .由于神经系统血脑屏障的存在 ,大分子肽链的给药途径是一个不可忽视的难题 .根据NGF的氨基酸序列及其晶体构象资料 ,选择CNBr在 9位Met处 ,胰蛋白酶在Arg或Lys处裂解 β NGF .经过SephadexG 5 0层析、DE 5 2纤维素离子交换层析和反相高压液相层析分离纯化后获得NGF活性片段 .氨基酸组成分析及氨基酸序列分析结果表明 ,此片段由 16肽GEFSVCDSVSVWVGDK与 14肽HWNSYCTTTHTFVK通过 1对二硫键连接而成 .8日龄鸡胚背根神经节生物活性分析表明 ,其最佳作用浓度为 0 0 0 1～ 0 1μg L .它的成功分离和鉴定为合成或表达高活性小分子神经营养物质奠定了关键而重要的基础 .     

Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12-day embryos and adult chicken also showed the activity. We partially purified this active substance from 12-day embryos, following procedures which included extraction at pH 3.5, CM-Sephadex C–50 ion exchange and Sephadex G-75 gel filtration. Judging from the dose-response analyses, the factor was purified by some hundred-fold when EE was used as the starting material. The activity was associated with a macro-molecular substance (MW > 300K daltons) at first, but the apparent molecular weight of the active substance was estimated to be between 16 and 20K daltons at the final step of the preparation. It promoted myoblast proliferation and delayed myotube formation, and was active for both avian and rat myoblasts.
Since bovine pituitary gland fibroblast growth factor (FGF) showed the same activity, the factor may be FGF-related.     

Changes in P2X<Subscript>3</Subscript> purinoceptors in sensory ganglia of the mouse during embryonic and postnatal development

The expression of the P2X₃ nucleotide receptor in embryonic day 14–18, postnatal day 1–14 and adult mouse sensory ganglia was examined using immunohistochemistry. Nearly all sensory neurons in dorsal root ganglia, trigeminal ganglia and nodose ganglia in embryos at embryonic day 14 expressed P2X₃ receptors, but after birth there was a gradual decline to about 50% of neurons showing positive immunostaining for P2X₃. In embryos there were only small neurons, while from postnatal day 7 both large and small neurons were present. Isolectin B₄ (IB₄)-positive neurons in dorsal, trigeminal and nodose ganglia did not appear until birth, but the numbers increased to about 50% by postnatal day 14 when a high proportion of IB₄-positive neurons were also positively labelled for the P2X₃ receptor. About 10% of neurons in dorsal, trigeminal and nodose ganglia were positive for calcitonin gene-related peptide in embryos, nearly all of which stained for P2X₃ receptors. This increased postnatally to about 35–40% in adults, although only a few colocalised with P2X₃ receptors. Neurofilament 200 was expressed in about 50% of neurons in trigeminal ganglia in the embryo, and this level persisted postnatally. All neurofilament 200-positive neurons stained for P2X₃ in embryonic dorsal root ganglia, trigeminal ganglia and nodose ganglia, but by adulthood this was significantly reduced. The neurons that were positive for calbindin in embryonic dorsal, trigeminal and nodose ganglia showed colocalisation with P2X₃ receptors, but few showed colocalisation postnatally.     

Isolation and characteristics of a nerve growth factor from the venom of the Central Asiatic viper Echis multisquamatus

The nerve growth factor (NGF) was isolated from the Echis multisquamatus venom by ultrafiltration on PM-10 filter, chromatography on TSK-55 gel, ion-exchange chromatography on CM-cellulose and gel filtration on Sephadex G-75. The protein exhibited a marked nerve growth activity within the concentration range of 10-15 ng/ml in cultures of chicken embryo spinal ganglia. The molecular mass of NGF is equal to 33,000-37,000 Da according to Sephadex G-75 gel filtration data; however, according to SDS electrophoresis data its Mr is 13,000 Da. Isoelectrofocusing data suggest that the pI of the isolated factor lies in the region of 7.0-7.2; sugar content is 1-2%.     

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.     

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

Summary The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.     

Brain-derived proteins that rescue spinal motoneurons from cell death in the chick embryo: Comparisons with target-derived and recombinant factors

Spinal motoneurons that normally die during early development can be rescued by a variety of purified growth or neurotrophic factors and target tissue extracts. There is also indirect evidence that brain or supraspinal afferent input may influence lumbar motoneuron survival during development and that this effect may be mediated by central nervous system–derived trophic agents. This report examines the biological and biochemical properties of motoneuron survival activity obtained from extracts of the embryonic chick brain. Treatment with an ammonium sulfate (25% to 75%) fraction of embryonic day 16 (E16) or E10 brain extracts rescued many spinal motoneurons that otherwise die during the normal period of cell death in vivo (E6 to E10). The same fractions also enhanced lumbar motoneuron survival following deafferentation. There were both similarities and differences between the active fractions derived from brain extracts (BEX) when compared with extracts derived from target muscles (MEX) or with purified neurotrophic factors. Survival activity from E10 BEX was as effective in promoting motoneuron survival as E10 MEX and more effective than astrocyte-conditioned media. Unlike MEX, the active fractions from BEX also rescued placode-derived nodose ganglion cells. In addition, unlike nerve growth factor and brain-derived neurotrophic factor, active BEX fractions did not rescue neural crest-derived dorsal root ganglion cells or sympathetic ganglion neurons. Interestingly, among many cranial motor and other brainstem nuclei examined, only the survival of motoneurons from the abducens nucleus was enhanced by BEX. Active proteins obtained from BEX were further separated by gel filtration chromatography and by preparative isolelectric focusing techniques. Activity was recovered in a basic (pI8) and an acidic (pI5) small molecular weight protein fraction (20 kD or less). The specific activity of the basic fraction was increased ×66 when compared with the specific activity of crude BEX, and the basic fraction had a slightly higher specific activity than the acidic fraction. The biological and biochemical properties of these fractions are discussed in the context of known neurotrophic factors and their effects on normal and lesion-induced motoneuron death during development. © 1995 John Wiley & Sons, Inc.     

The development of the neurons of the glossopharyngeal (IX) and vagal (X) sensory ganglia in chick embryos

The timetable of cell generation, neuronal death and neuron numbers in the fused proximal glossopharyngeal (IX) and vagal (X) ganglion and distal IX and X ganglia were studied in normal and nerve growth factor (NGF) treated chick embryos. 3H-thymidine was injected between the 3rd and 7th days of incubation and embryos sacrificed on the 11th day. Neurons in the distal IX and X ganglia were generated between the 2nd and 5th days of incubation, the peak mitotic activity occurring on the 4th and 3rd days, respectively. Neurons of the proximal IX and X ganglion were generated between the 4th and 7th days, with maximum neuron generation on the 5th day of incubation. Counts of neurons in the 3 ganglia between the 5th and 18th days of incubation showed a maximum of 22,000 on the 8th day in the proximal IX and X ganglion and this decreased to 12,000 by the 13th day. In the distal IX ganglion, the neuron number decreased by 44% from 4,500 on the 6th day to 2,500 by the 11th day. A similar decrease of 43% was found in the distal X ganglion, the neuron number falling from 11,500 on the 7th day to 6,500 by the 11th day of incubation. Neuronal cell death in these ganglia extended from the 5th to the 12th day of incubation, maximum cell death occurring at or after the cessation of mitotic activity. NGF administration from the 5th to the 11th day of incubation did not have a measurable effect on the neurons of proximal IX and X and distal IX ganglia, but increased neuronal survival by 30% in the distal X ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of receptors for alpha-bungarotoxin in chick embryo sympathetic ganglion neurons in vitro

The development of receptors for α-bungarotoxin was examined in neurons in dissociated cultures of cells derived from chick embryo sympathetic ganglia. Neurons from 12 day embryos showed a marked increase in receptor numbers per cell over 3–4 days in culture. The increase was less marked in neurons from 14 day embryos and absent in 19 day embryos. The incidence of cholinergic synapses in cultures from 12 day and 19 day embryos was also examined. Evidence for synapse formation was found only in cultures from older embryos.     

Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure,and quantitative determinations of catecholamines and somatostatin

Summary The ultrastructural and transmitter development of lumbar sympathetic ganglia was studied in embryonic day-6 through-18 chick embryos. At embryonic day 6, ganglia are populated by two morphologically distinct types of neuronal cells and Schwann cell precursors. The neuronal populations basically comprise a granule-containing cell and a developing principal neuron. Granule-containing cells have, an irregularly shaped or oval nucleus with small clumps of chromatin attached to the inner nuclear membrane and numerous large (up to 300 nm) membrane-limited granules. Developing principal neurons display a more rounded vesicular nucleus with evenly distributed chromatin, prominent nucleoli, more developed areas of Golgi complexes, and rough endoplasmic reticulum and large dense-core vesicles up to 120 nm in diameter. There are granule-containing cells with fewer and smaller granules which still display the nucleus typical for granule-containing cells. These granule-containing cells may develop toward developing principal neurons or the resting state of granule-containing cells found in older ganglia. Both granule-containing cells and developing principal neurons proliferate and can undergo degeneration. At embryonic day 9 there are far more developing principal neurons than granule-containing cells. Most granule-containing cells have very few granules. Mitotic figures and signs of cell degeneration are still apparent. Synapse-like terminals are found on both developing principal neurons and granule-containing cells. Ganglionic development from embryonic day 11 through 18 comprises extensive maturation of developing principal neurons and a numerical decline of granule-containing cells. Some granule-containing cells with very few and small granules still persist at embryonic day 18. The mean catecholamine content per neuron increases from 0.044 femtomol at embryonic day 7 to 0.22 femtomol at embryonic day 15. Concomitantly, there is a more than 6-fold increase in tyrosine hydroxylase activity. Adrenaline has a 14% share in total catecholamines at embryonic day 15. Somatostatin levels are relatively high at embryonic day 7 (1.82 attomol per neuron) and are 10-fold reduced by embryonic day 15. Our results suggest the presence of two morphologically distinct sympathetic neuronal precursors at embryonic day 6: one with a binary choice to become a principal neuron or to die, the other one, a granule-containing cell, which alternatively may develop into a principal neuron, acquire a resting state or die.     

The role of substance P-containing fibers in sympathetic ganglia: Effect of capsaicin

Capsaicin was given subcutaneously to guinea pigs and the effect on substance P-immunoreactive (SP-I) fibers in the celiac/superior mesenteric and inferior mesenteric ganglia was observed at 2 day and 8–10 day intervals. Capsaicin (125 mg) treatment led to almost total disappearance of SP-I fibers from all areas examined in both short- and long-term animals. This effect applied equally to the dense network of varicose SP-I fibers and to basket-like SP-I contacts with principal ganglionic neurons. The effect of capsaicin on SP-I fibers in the mesenteric ganglia provides a strong indication that these fibers represent a homogenous population of visceral sensory afferents. This is supported by other lines of anatomical evidence in the literature. Taken together with studies that have shown axodendritic contact of SP-I terminals on principal ganglionic neurons and neuro-modulatory effects of SP on these neurons, it may be hypothesized that SP-I fibers in the mesenteric ganglia represent collaterals of visceral sensory afferents forming a subspinal feedback arc.     

Effects of ageing on peroxidase activity and localization in radish (Raphanus sativus L.) seeds

Peroxidase activity was assayed in crude extracts of integument, cotyledons and embryo axis of radish seeds, deteriorated under accelerated ageing conditions. Over five days of ageing, in which germination decreased from 100 to 52%, the enzyme activity in integument was higher than that in other seed parts, increasing in the first days of ageing and then decreasing sharply in extremely aged seeds. Polyacrylamide gel electrophoresis analysis showed four peroxidase isoenzymes with MM of 98, 52.5, 32.8 and 29.5 kDa in the embryo axis of unaged seeds, and only the 32.8 and 29.5 kDa MM isoforms in the integument and cotyledons. In these parts of the seed, only the 29.5 kDa MM isoenzyme increased in activity in early days of ageing and decreased there-after. In the embryo axis, the 29.5 kDa MM isoenzyme activity increased slowly in the first day of ageing, while the 98 and 52.5 kDa MM isoenzyme activities disappeared. A cytochemical localization of peroxidase activity in the various tissues showed that main differences between unaged and extremely aged seeds occurred in the embryo axis.     

A large proportion of afferent neurons innervating the uterine cervix of the cat contain VIP and other neuropeptides

Summary Axonal tracing techniques were used in combination with immunohistochemistry to examine the distribution of neuropeptides in afferent pathways from the uterine cervix of the cat. Primary afferent neurons innervating the uterine cervix were identified by axonal transport of the dye, fast blue, injected into the cervix. Fifteen to twenty-five days after the injection, dorsal root ganglia (L1–S3) were removed and incubated for 48–72 h in culture medium containing colchicine to increase the levels of peptides. Calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), leucine-enkephalin (LENK), somatostatin, substance P and vasoactive intenstinal polypeptide (VIP) were identified by use of indirect immunohistochemical techniques. Eighty-four percent of uterine cervix afferent neurons were identified in the sacral dorsal root ganglia (S1–S3), and 16% in the middle lumbar dorsal root ganglia (L3–L4). In sacral dorsal root ganglia, VIP was present in the highest percentage of dye-labeled cells (71%), CGRP in 42%, and substance P in 18% of the cells. CCK and LENK were present in 13% of the cells. In lumbar dorsal root ganglia, CGRP (51%) was most prominent peptide followed by VIP (34%), substance P (28%), LENK (17%) and CCK (13%). Somatostatin was present in the ganglia but did not occur in dye-labeled neurons. In conclusion, the uterine cervix of the cat receives a prominent VIP-and CGRP-containing afferent innervation. The percentage of neurons containing VIP is three to five times higher than the percentage of these neurons in afferent pathways to other pelvic organs. These observations coupled with the results of physiological studies suggest that VIP is an important transmitter in afferent pathways from the cervix.     

Presence of Somatostatin, Enkephalins, and Substance P-Like Peptides in Cultured Neurons from Embryonic Chick Cerebral Hemispheres

The presence of peptides in pure cultures of neurons from 8-day-old chick embryo cerebral hemispheres has been investigated by means of specific radioimmunoassays and chromatographic purification. Somatostatin, Met-enkephalin, Leu-enkephalin, and substance P immunoreactive substances have been detected in 8-day-old cultures grown in serum-free culture medium. The peptides were present in the cellular extracts, as well as in the culture medium extracts. beta-Endorphin, thyroliberin, luteinizing hormone-releasing hormone, and ACTH could not be detected. The largest amount was accounted by somatostatin (48 +/- 2 ng/mg protein). Some 60% of the somatostatin-immunoreactive material was found in the culture medium. Met-enkephalin, Leu-enkephalin, and substance P were present at lower concentrations: 1.61 +/- 0.27, 0.24 +/- 0.02, and 0.14 +/- 0.005 ng/mg protein, respectively. The identities of somatostatin- and enkephalin-immunoreactive materials were confirmed by high pressure liquid chromatography. The findings suggest that cultured neurons that express dopaminergic and GABAergic properties contain peptides similar, if not identical, to somatostatin, Met-enkephalin, Leu-enkephalin, and substance P.     

Nerve growth factor-mediated induction of tyrosine hydroxylase in rat superior cervical ganglia in vitro.

Exposure of rat sympathetic ganglia to 3 microgram/ml of 2.5 S nerve growth factor (NGF) resulted in a 100% increase in tyrosine hydroxylase activity within 48 h. Pulselabeling of proteins with [3H]leucine, followed by immunoprecipitation with antibodies to tyrosine hydorxylase and isolation of the precipitated enzyme by gel electrophoresis, demonstrated that the increase in tyrosine hydroxylase activity was due to enhanced de novo synthesis. The incorporation of [3H]leucine into tyrosine hydroxylase was increased by 150% compared to a 17% increase in total protein synthesis, which was not statistically significant. The fact that the half-life of pulse-labeled tyrosine hydroxylase was the same for NGF-treated and control organ cultures of superior cervical ganglia excludes the possibility that enhanced tyrosine hydroxylase labeling by NGF is due to decreased degradation. We conclude that, without modulatory factors which play a role in vivo, NGF can enhance the synthesis of tyrosine hydroxylase in sympathetic ganglia in vitro, provided organ culture conditions which permit optimal survival of adrenergic neurons are selected.