Chick Embryo Brain Research Articles

Organic and inogranic lead inhibit neurite growth in vertebrate and invertebrate neurons in culture

Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl2), or organic lead (triethyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 270 microM total lead, approximately 70 nM free Pb2+) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.24 microM) and the mean neurite length (extrapolated IC50 = 3.6 microM) but did not reduce the number of neurites per cell. In Lymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 13 microM total lead; approximately 10 nM free Pb2+). Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.4 microM) and exerted significant toxicity at 0.2 microM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechanisms of action are different.

Embryonal Development of Arginine Vasotocin/Mesotocin Gene Expression in the Chicken Brain

Abstract An ontogenic series of chick embryo brains (4, 6, 9,12,14,16 and 18 days of incubation, hatching day: 21) was coronally or saggitally sectioned and investigated for expression of the arginine vasotocin (AVT)/mesotocin (MT) gene. To this end a 39mer oligonucleotide recognizing the AVT/MT encoding sequence of their respective mRNAs was constructed employing optimized codon usage and was used for in situ hybridization. AVT/MT mRNA-expressing neurons were first detected on embryonal day (E) 6 adjacent to the third ventricle. By E9 the periventricular nucleus had expanded in size but the hybridization signal was weaker. These results suggest a migration of cells in all directions away from the third ventricle into the diencephalon; some perikarya were even observed at the lateral pial surface above the optic chiasm. By E12, brain differentiation had advanced to distinct hypothalamo-neurohypophyseal nuclei (supraoptic and paraventricular nuclei) as well as to accessory groups expressing AVT/MT mRNA. Two cell types were then distinguishable in the paraventricular nucleus; the specific mRNA was expressed either as a weak or a strong hybridization signal. Comparison with other studies suggested that differentiation had almost attained adult level though cell growth and differentiation of supraoptic and paraventricular nuclei still occurred. This was complete by E18 when individual cells were larger and the intensity of the hybridization signal became very strong. RNase pretreatment depressed the signal by 100%. Northern blot hybridization of RNA extracted from newly hatched chicks verified the specificity of the probe. A single band was evident, indicating an mRNA of approximately 700 bases, which is only found in hypothalamus and not in muscle, liver or extra-hypothalamic brain, and corresponds in size closely with mammalian vasopressin mRNA. The data demonstrate a very early development (E6) of neurohypophyseal hormone gene expression in the chick embryo brain with important differentiation around E9. At E12 magnocellular neuronal arrangement resembled adult neurons but the gene expression signal increased in intensity until E18.

O 2 effect on composition of chick embryonic heart and brain

Heart ventricles from chick embryos incubated in 60% O 2 (hyperoxia) on the 16 th through the 18 th days of incubation were 21% heavier than those from control embryos maintained in 21% O 2 (normoxia). Heart ventricles from embryos incubated in 15% O 2 (hypoxia) were 8% lighter than controls. Changes in ventricular weight were accompanied by proportional changes in protein content (21% more in hyperoxic ventricles; 8% less in hypoxic ventricles). Ventricular tissue DNA content showed a significant increase in hyperoxia. Tissue protein/DNA ratios were significantly higher in hyperoxia and lower in hypoxia. These data suggest that increased O 2 availability led to hypertrophy of chick embryo ventricular cells and an increase in the level of DNA synthesis. Cytochrome oxidase activity per mg DNA was 15–25% higher in hyperoxic ventricles than in hypoxic ventricles. This result is consistent with our previous findings that alterations in O 2 availability affect the O 2 consumption rate of the chick embryo in ovo, and it provides direct evidence that a phenomenon repeatedly observed in vitro is of importance in vivo. In contrast to the heart, O 2 availability did not affect the wet weight, protein or DNA contents, or cytochrome oxidase activity of the chick embryo brain.

Effect of phospholipids on the activity of sialosyl lactosylceramide (GM3): N-acetylgalactosaminyl transferase from chick embryo brain

A preparation of UDP-GalNAc:sialosyl lactosylceramide N-acetylgalactosaminyl transferase (E.C. 2.4.1.92) obtained from 14-day chick embryo brain was delipidated partially by treatment with cold acetone in the presence of varying amounts of sodium dodecyl sulphate (SDS). The lipid content and the enzyme activity of the preparation decreased as the concentration of SDS increased. At 0.3% SDS the lipid content was about 30% and the enzyme activity about 15% of the original. The activity could be restored up to 60% of the original by added phospholipids, provided the removal of endogenous lipids did not exceed 70%. Phospholipids with different composition showed different abilities to restore the enzyme activity. Among phosphatidylcholines the decreasing order of effectivity was dilauroyl----dimiristoyl----dipalmitoyl----distearoyl-choline. Dimiristoyl phosphatidylcholine, dimiristoyl phosphatidylglycerol and dipalmitoyl phosphatidylglycerol activated the enzyme more effectively than dimiristoyl phosphatidyl ethanolamine, dimiristoyl phosphatidic acid or brain phosphatidylserine. No correlation was found between the activating ability and the charge in the polar head group of the lipid added. Addition of dilauroyl phosphatidylcholine to the delipidated preparation increased about 5-fold the Vmax without affecting the apparent Km for both the donor nucleotide and acceptor glycolipid. The data suggest that the lipid composition of the enzyme environment constitutes a potential level of regulation of the activity of this key enzyme of ganglioside biosynthesis.

A developmentally regulated chicken neuronal protein associated with the cortical cytoskeleton

Monoclonal antibody 3D5 recognizes a single component of the neuronal membrane skeleton isolated from the chicken embryo brain. The 3D5 antigen is highly enriched in the CNS, and smaller amounts are found in the PNS. It is also present in non-neural tissues, but this is due to peripheral innervation. The biochemical and molecular properties of the 3D5 antigen are very similar to those of the previously described mammalian protein B-50 (Zwiers et al., 1985)/GAP 43 (Jacobson et al., 1986)/pp46 (Ellis et al., 1985)/F1 (Chan et al., 1986), and include anomalous SDS gel migration, acidic isoelectric point, and extraction from the cytoplasmic side of the plasma membrane only under extremely alkaline conditions. The 3D5 antigen is also developmentally regulated, with maximum expression in brain occurring at E14-E16, after which levels decrease approximately 4-fold in the adult. Immunofluorescence staining of cultured neurons shows that the 3D5 antigen is located in all parts of the cell but is particularly enriched in the growth cone and the growth cone filopodia. As the 3D5 antigen is enriched in the membrane skeleton, we suggest that this protein is involved in an association between the actin-containing cytoskeleton and the plasma membrane.

Correlation of neuropathy target esterase activity with specific tritiated di-isopropyl phosphorofluoridate-labelled proteins.

Neuropathy target esterase (NTE) is a membrane-bound carboxylesterase activity that has been proposed as the target site for initiation of organophosphate-induced delayed neuropathy. This activity is identified by its resistance to treatment with Paraoxon and sensitivity to co-incubation with Paraoxon and Mipafox. Sucrose-density-gradient centrifugation of membrane-associated proteins isolated from chick-embryo brains identified three proteins, Mr 161,000, 116,500 and 103,000, that were labelled with [3H]di-isopropyl phosphorofluoridate in an NTE-like manner and that co-migrated with NTE. The 161,000-Mr and 116,500-Mr proteins were identified in both adult and embryo brain. One or both of these proteins may therefore contribute to the activity defined as NTE. In addition, a 61,000-Mr protein was identified that does not comigrate with NTE, but that was labelled with [3H]di-isopropyl phosphorofluoridate in a Paraoxon-resistant and Mipafox-sensitive manner. The effect of Mipafox on labelling, however, was reversibly blocked by co-incubation with Paraoxon. This protein, therefore, is not NTE, but has the necessary inhibitor-sensitivity to be the target site for organophosphate-induced delayed neuropathy.

A low-molecular weight chick brain-derived growth factor is mitogenic for cultured astroglia from the chick embryo

Immunohistochemically defined astrocytes from the 10-day chick embryo were stimulated to incorporate increased levels of [ 3H]thymidine when a low-molecular weight peptide growth factor, chick brain-derived growth factor (CBGF), was added to the cultures. Treatment of these GFAP-positive astrocytes with 10 ng/ml CBGF in medium supplemented with 1% fetal bovine serum resulted in a 3.5–4-fold increase in [ 3H]thymidine incorporation when compared to astrocytes cultured in defined medium supplemented with 1% serum alone. CBGF had no effect on the survival, proliferation or differentiation of a number of other cell types from the 10-day chick embryo brain, including neurons and meningeal fibroblasts. CBGF was also ineffective as a mitogen for chick embryo skeletal muscle myoblasts, primary mouse embryo fibroblasts and one murine teratocarcinoma-derived cell line (STO). We suggest that CBGF might act as a mitogenic signal for astroglia during central nervous system development and repair.

Distribution in cesium chloride gradients of proteoglycans of chick embryo brain and characterization of a large aggregating proteoglycan

Proteoglycans were extracted from 14-day chick embryo brains, which had been labelled in vitro with [ 35S]sulfate or 3H-labelled amino acids. 4.0 M guanidinium chloride (containing proteinase inhibitors) extracted 94% of the 35S-labelled glycoconjugates. Following cesium chloride equilibrium centrifugation, the proteoglycans in each fraction were characterized by chromatography on Sepharose CL-2B. The most dense fraction (D1), which contained no detectable non-proteoglycan proteins, contained a large, aggregating chondroitin sulfate proteoglycan in addition to small chondroitin sulfate and heparan sulfate proteoglycans. The less dense fractions (D2–D6) contained both small chondroitin sulfate and heparan sulfate proteoglycans. Removal of hyaluronate from the D1 sample by digestion with Streptomyces hyaluronidase in the presence of proteinase inhibitors showed that aggregation of the large chondroitin sulfate proteoglycan is hyaluronate-dependent. Aggregation was restored by re-addition of hyaluronate. Reduction and alkylation, which blocked aggregation of a cartilage A1 proteoglycan, did not interfere with aggregation of the large brain proteoglycan.

Development of the amino acid pools in chick embryo brain, heart, and eye: taurine, valine, glutamine, and phosphoethanolamine.

The redistribution of valine, from the nonrenewable yolk supply into excitable tissues, was studied during the first 15 days of chick embryogenesis. Valine levels in the extraembryonic circulation (the vitelline plexus) peak between days 7-9 (E7-9) and then decline steeply. In their first phase of differentiation (E2-E4), all embryonic tissues contain more valine than the blood plexus. From E4 to E7, the heart and brain exhibit initially a rapid fall in valine, but from day 7 on the decrease becomes more gradual. The eye during the same period reaches an equilibrium with circulating valine; as these levels fall from E9 to E15, the eye retains the valine that accumulated. Against this pattern of change, characteristic for an essential amino acid during embryogenesis, glutamine levels are at any time from two- to threefold higher than valine in all tissues. In the circulation, this ratio remains constant throughout the 15 days of embryonic development. Eye glutamine, higher on day 4, by E7 has entered into an equilibrium with glutamine in the plexus. A steady but two times higher glutamine level is maintained in the heart, although during the later stages of development it gradually tends to approach the plexus content. In sharp contrast, starting on E7 and accelerating on E9, a large increase of glutamine relative to valine or other essential amino acids is seen in developing brain tissue. This appears typical for most metabolic amino acids, suggesting that by days 9-10 the essential amino acid supply in the brain is being exhausted.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxic effects of phencyclidine on developing chick embryo brain

We studied the effects of Phencyclidine (PCP, Angel Dust) on the developing chick embryo brain. In Group-1, the eggs were injected with PCP on the 7th day of incubation and the embryo brains were studied on the 10th day. In Group-2, eggs were injected twice; first on the 7th day and then 10th day of incubation. Group-2 brains were then studied on the 16th day of incubation. PCP significantly depressed the development of embryo brains. Cerebral hemisphere weight, total protein and total DNA were significantly lowe on day 10 of incubation in Group-1. Similar results were observed in Group-2. Concomitantly, the concentration of brain serotonin at day 10 was also significantly reduced when PCP was injected into the eggs on the 7th day of incubation. Since serotonin has been reported to influence development of the chick embryo brain, the present finding of the effect of PCP on brain development might be a secondary phenomenon. The possible implications of the effects of PCP on human brain development are also discussed.

Characterization of acidic and basic fibroblast growth factors in brain, retina and vitreous chick embryo.

We have purified acidic and basic fibroblast growth factors (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these growth factors.

GABA receptors induced in Xenopus oocytes by chick brain mRNA: evaluation of TBPS as a use-dependent channel-blocker

Sensitivity to GABA was induced in Xenopus laevis oocytes by injection of poly(A) + mRNA extracted from 19-day chick embryo brain. The effect of the convulsant drug t-butylbicyclophosphorothionate (TBPS) was studied on the responses to bath-applied GABA using voltage-clamp techniques. TBPS reversibly inhibited the GABA-evoked current ( I GABA) in a dose-dependent manner; however, the chloride currents evoked by carbachol or serotonin, or the spontaneous chloride fluctuations, were unaffected. The onset of the block by TBPS was faster in the presence of GABA. The recovery from the block after TBPS wash-out was also agonist-stimulated. At the steady state block, TBPS showed a mixed type of inhibition: increasing the GABA concentration decreased but failed to abolish completely the inhibition by TBPS. The TBPS block was independent of the direction of the chloride flow: both inward and outward I GABA were blocked. The time course of the decay of I GABA was markedly changed in the presence of TBPS: above 40 μM GABA, this time course showed essentially two exponentials and TBPS abolished only the fast component, whereas at a low concentration (⩽4 μM), I GABA was relatively constant and uniformly reduced by TBPS. It is suggested that TBPS may stabilize a closed form of the liganded receptor-channel complex.

Differential maturation of ? and ? opioid receptors in the chick embryonic brain

The developmental profiles of the binding of mu and delta opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [3H]dihyromorphine was used as a mu ligand and with 5 X 10(-7) M levorphanol for non-specific binding, and [3H](D-Ala2-D-Leu5)-enkephalin was used as a delta with 5 X 10(-7) M (D-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both mu and delta opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity mu sites during early embryogenesis but only one delta site. By 18 days of embryonic age, only one mu site remained. This developmental change is interpreted as a transitory state of the receptor to the adult mu pattern. The presence of only one delta site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the mu receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.

O-80 - The effect of ethanol on the incorporation of inositol and choline into phospholipids in the brain of chick embryo

O-80 - The effect of ethanol on the incorporation of inositol and choline into phospholipids in the brain of chick embryo

Growth promoting effect of head activator in cultured chick embryo brain cells.

To investigate the regulatory role of head activator (HA) and its synthetic analogue, (Arg1,Phe5)-HA(AHA) on brain cell growth, we measured serial uptakes of [3H]thymidine, [3H]uridine and [3H]leucine and changes in cyclic AMP content in cultured chick embryo brain cells. HA stimulated all of these uptakes at a concentration of 10(-10) M, while 10(-9) M AHA suppressed them. The stimulatory effect of HA on [3H]thymidine uptake was observed after 4 h of the treatment, reached a maximum of 200% of the initial value at 8 h and declined to the pre-treatment level at 14 h. [3H]uridine uptake began to increase after 6 h of HA treatment, and the effect lasted for 4 h. Increase in [3H]leucine followed after 12 h and sustained for 4 h. Prior to the initiation of HA stimulation, cyclic AMP also began to rise, reaching 170% of the pre-treatment level at 6 h. In contrast, depression of [3H]thymidine uptake by AHA was noted at 6 h and continued for 8 h. Uptake of [3H]uridine and [3H]leucine showed similar tendency. Cyclic AMP in AHA-treated cells at 6 h was significantly lower than that in non-treated cells. These results indicate that HA stimulates DNA, RNA and protein synthesis in an early stage of growing brain cells, in which cAMP may be involved as a regulator of nerve cell growth.

Improved potency test for live avian encephalomyelitis vaccines

Potency tests were carried out on live avian encephalomyelitis virus (AEV) vaccines. Vaccines were titrated in chick embryo brain cell cultures using an indirect fluorescent antibody test just before vaccination. Groups of chickens were inoculated orally with graded doses of each vaccine. After three weeks serum was taken from the chickens and examined for antibodies to AEV by indirect enzyme-linked immunoassay (ELISA). The chickens were then challenged by intracerebral inoculation of virulent AEV and monitored for signs attributable to clinical AE. The results showed a relationship between virus content, protection and antibody development. It is recommended that serological evaluation using ELISA replace the challenge step in potency tests for live AEV vaccines.

Purification of a membrane protein distributed in a topographic gradient in chicken retina.

Antigenic molecules termed TOP, which are distributed in a dorsal greater than ventral concentration gradient in chicken retina, are expressed early in development (by 48 hr after fertilization) in the optic cup of chicken embryos and continue to be expressed in retina thereafter. 35S-labeled-TOP-antibody complexes were purified by protein A-Sepharose column chromatography and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. TOP also was purified from dorsal retina by anti-TOP IgG-Affi-Gel 10 affinity column chromatography. In both cases, one major band of protein at Mr approximately 47,000 was obtained. A protein of Mr approximately 47,000 also was purified from chicken embryo brain. Cultured cells dissociated from 8-day chicken embryo retinas accumulated the amount of TOP expected of cells in the intact retina, depending on the position of the cells in the retina. TOP accumulations by cells dissociated from dorsal or ventral retina, mixed in different proportions, and cocultured were additive. These results show that TOP is a protein, that the gradient of TOP is established early in development, and that perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusable molecules or on maintenance of interactions between cells.

Isofenphos and an in vitro activation assay for delayed neuropathic potential

Organophosphorus compounds (OPs) that cause organophosphorus ester-induced delayed neuropathy (OPIDN) generally inhibit neurotoxic esterase (NTE). However, the assay itself, when conducted in vitro, misses OPs that are activated into OPIDN-causing agents in the body. A preparation of liver mixed-function oxidases and brain NTE was used to rapidly detect activations of OPs. The compounds (0.1 m m or less) to be tested were incubated with microsomes isolated from livers of phenobarbital-treated chick embryos ( P-450 content averaged 1.81 ± 0.27 nmol/mg protein, x ± SD, N = 5 ) and NTE (average of 13.8 nmol/min/mg protein) from untreated chick embryo brains. The NTE was separated by calcium precipitation and its activity assayed as usual. The low inhibitions of NTE of compounds that were not neurotoxic (parathion, Diazinon) did not increase in the presence of NADPH; inhibitions of NTE of compounds that required activation (leptophos, S,S,S-tri- n-butyl phosphorotrithioate, and tri- o-cresyl phosphate) greatly increased with NADPH. Both the recently indentified neuropathic OP isofenphos (IFP) and its oxon required activation to inhibit NTE (inhibitions of 20 and 80%, respectively). Evidence is presented that the possible neuropathic metabolite is des- N-isopropyl IFP oxon.

Detection of avian encephalomyelitis virus

Methods for the detection of two strains of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures and chickens were compared. It was found that the agar gel precipitin test (AGPT) and the enzyme-linked immunosorbent assay (ELISA) carried out on the serum of inoculated chickens were more sensitive than either the indirect fluorescent antibody test in cell cultures or the detection of clinical signs in chicks. On the basis of results obtained in this experiment the effects were then determined of routes and time of inoculation of chickens on the detection of AEV. It was found that birds infected at two weeks old produced higher antibody titres than one-day-old birds and the AGPT and ELISA detected comparable levels of antibody in them. It was recommended that the tests to detect the presence of AEV as a contaminant of vaccines be replaced by a serological test carried out on chicks inoculated intramuscularly at two weeks old.

Corticosterone accelerates the development of electrical properties in chick embryo brain cells in culture

Corticosterone accelerates the development of electrical properties in chick embryo brain cells in culture