Chicken Brain Tissue Research Articles

Off-center spherical model for dosimetry calculations in chick brain tissue.

This paper presents calculations for the electric field and absorbed power density distribution in chick brain tissue inside a test tube, using an off-center spherical model. It is shown that the off-center spherical model overcomes many of the limitations of the concentric spherical model, and permits a more realistic modeling of the brain tissue as it sits in the bottom of the test tube surrounded by buffer solution. The effect of the unequal amount of buffer solution above the upper and below the lower surfaces of the brain is analyzed. The field distribution is obtained in terms of a rapidly converging series of zonal harmonics. A method that permits the expansion of spherical harmonics about an off-center origin in terms of spherical harmonics at the origin is developed to calculate in closed form the electric field distribution. Numerical results are presented for the absorbed power density distribution at a carrier frequency of 147 MHz. It is shown that the absorbed power density increases toward the bottom of the brain surface. Scaling relations are developed by keeping the electric field intensity in the brain tissue the same at two different frequencies. Scaling relations inside, as well as outside, the brain surface are given. The scaling relation distribution is calculated as a function of position, and compared to the scaling relations obtained in the concentric spherical model. It is shown that the off-center spherical model yields scaling ratios in the brain tissue that lie between the extreme values predicted by the concentric and isolated spherical models.

Effects of ELF (1-120 Hz) and modulated (50 Hz) RF fields on the efflux of calcium ions from brain tissue in vitro.

We have previously shown that 16-Hz, sinusoidal electromagnetic fields can cause enhanced efflux of calcium ions from chick brain tissue, in vitro, in two intensity regions centered on 6 and 40 Vp-p/m. Alternatively, 1-Hz and 30-Hz fields at 40 Vp-p/m did not cause enhanced efflux. We now demonstrate that although there is no enhanced efflux associated with a 42-Hz field at 30, 40, 50, or 60 Vp-p/m, a 45-Hz field causes enhanced efflux in an intensity range around 40 Vp-p/m that is essentially identical to the response observed for 16-Hz fields. Fields at 50 Hz induce enhanced efflux in a narrower intensity region between 45 and 50 Vp-p/m, while radiofrequency carrier waves, amplitude modulated at 50 Hz, also display enhanced efflux over a narrow power density range. Electromagnetic fields at 60 Hz cause enhanced efflux only at 35 and 40 Vp-p/m, intensities slightly lower than those that are effective at 50 Hz. Finally, exposures over a series of frequencies at 42.5 Vp-p/m reveal two frequency regions that elicit enhanced efflux--one centered on 15 Hz, the other extending from 45 to 105 Hz.

Cell-substratum adhesion in embryonic chick central nervous system is mediated by a 170,000-mol-wt neural-specific polypeptide.

Embryonal chick neural retina cells release into the culture medium a complex of proteins and glycosaminoglycans, termed adherons, that promote cell to substratum adhesion. A monoclonal antibody (C1H3) blocks adheron-mediated cell to substratum adhesion and specifically binds to a 170,000-mol-wt protein present in retinal adherons (Cole, G.J., and L. Glaser, 1984, J. Biol. Chem., 259:4031-4034). The 170,000-mol-wt protein also can be identified in embryonic chick brain and peripheral nervous tissue. In the neural retina, C1H3 also binds to a second antigen with a molecular weight of 140,000 that is absent in the brain. Embryonic brain, therefore, provides a source for the immunopurification of the 170,000-mol-wt protein. Brain adherons also contain the 170,000-mol-wt protein, and cell to substratum adhesion mediated by these adherons is blocked by the C1H3 monoclonal antibody. The 170,000-mol-wt protein in the brain is therefore functionally identical to that in the retina. To demonstrate that adheron-mediated cell to substratum adhesion is caused by cell binding to the 170,000-mol-wt protein, we showed that (a) protease digestion, but not glycosaminoglycan hydrolase digestion of adherons, blocked their ability to bind cells to substratum; (b) the immunopurified 170,000-mol-wt protein blocks adheron-mediated cell to substratum adhesion; and (c) cells can bind to immunopurified 170,000-mol-wt protein bound to glass surfaces.

Sulfation and phosphorylation of the neural cell adhesion molecule, N-CAM.

Embryonic chicken brain tissue cultured in media containing 35S-labeled sulfate or 32P-labeled phosphate incorporated 35S or 32P into the neural cell adhesion molecule (N-CAM). The 35S label was located in asparagine-linked carbohydrates on both glycopeptides (molecular weights, 170,000 and 140,000) but not in the sialic acid. The 32P label was detected in phosphoamino acids in the carboxyl-terminal third of both polypeptides, but the ratio of phosphoserine to phosphothreonine differed in the two species. The sulfated saccharides and phosphoamino acids may provide additional sites for functional control of N-CAM.

Developmental and biochemical analysis of chick brain tubulin heterogeneity.

Tubulin, isolated from brain tissue of chicks at different stages during late embryonic and early post-hatched development by ion-exchange chromatography and by in vitro microtubule reassembly, was analyzed by high-resolution isoelectric focusing and by two-dimensional polyacrylamide gel electrophoresis. Similar results were obtained with tubulins purified by the two methods. Sixteen isoelectric species of tubulin that differ in apparent net charge under denaturing conditions were detected by isoelectric focusing. By two-dimensional polyacrylamide gel electrophoresis, the chick brain tubulins were resolved into at least seven forms of alpha and 10 forms of beta tubulin. The number and relative proportions of the multiple brain tubulins were modulated during development. Since there are only four alpha tubulin and four beta tubulin genes in chickens, posttranslational modification of the tubulins must play a prominent role in the heterogeneity. Analysis of isotubulin distributions through cycles of microtubule assembly and disassembly indicated that the tubulins differ very little, if at all, in their capacity to assemble into microtubules. Therefore, the chemical differences that distinguish the multiple tubulins have very little structural impact on the protein surface areas involved in microtubule formation. Partial fractionation of the multiple tubulins during ion-exchange chromatography was observed, suggesting that it may be possible to isolate individual native tubulin variants for biochemical studies.

Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides.

beta-Tubulin subunits isolated from chicken brain tissue and erythrocytes are distinguishable as unique biochemical species by electrophoretic and peptide mapping procedures. 1) The subunits of beta-tubulin exhibit major differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels that vary according to the pH and ionic strength of the gel. 2) The isoelectric points of urea-denatured beta subunits from brain tissue and erythrocytes are pH 5.1 and 5.4, respectively, whereas those of both alpha subunits are approximately pH 5.2.3) Two-dimensional peptide maps prepared with alpha-chymotrypsin or V8 protease show that alpha-tubulin peptides are indistinguishable, whereas beta-tubulin peptides are very different. Only one-third of the 15 major tyrosine-containing beta-tubulin peptides prepared with alpha-chymotrypsin are common to both beta-tubulin species. The data indicate that the beta-tubulin subunits of brain tissue and erythrocytes are biochemically distinct and may be different gene products. The presence of tubulin variants in brain tissue and erythrocytes may indicate special requirements for microtubule assembly and function in different cell types.

Implication of iron in seizure syndrome of mutant chicks ( Gallus domesticus)

1. 1. An investigation was made of systemic iron in paroxysmal ( px) chicks. 2. 2. Serum transferrin levels in px chicks are greatly increased ( P < 0.05) over control levels, but iron-binding capacity is decreased ( P < 0.05). 3. 3. The delayed (by 10 days) increase in px hemoglobin ( P < 0.01) may be due to the general debilitation seen in px syndrome. 4. 4. An attempt to determine brain iron showed no deposits comparable to mammalian brain iron distribution, but this was consistent with inability of others to locate iron in adult chicken brain tissue.

Effects of ELF Fields on Calcium-Ion Efflux from Brain Tissue in Vitro

It has been previously demonstrated that carrier waves of 50 and 147 MHz, when sinusoidally amplitude modulated at 16 Hz (ELF), can cause enhanced efflux of radiolabeled calcium ions from chick brain tissue in vitro. This phenomenon occurs only when the samples are exposed to specific intensity ranges of the carrier wave. Unmodulated carrier waves do not affect the ion efflux. Since the ELF signal must be demodulated from the carrier wave to be effective, a study of the efflux ehnancement due to the ELF signal alone may lead to an identification of the site of demodulation, as well as provide clues to the underlying mechanism. We report here that 16-Hz sinusoidal fields in the absence of a carrier wave can alter the efflux rate of calcium ions. The results show a frequency-dependent, field-induced enhancement of calcium-ion efflux within the ranges 5 to 7.5 V/m and 35 to 50 V/m (peak-to-peak incident field in air) with no enhancement within the ranges 1 to 2, 10 to 30, and 60 to 70 V/m.

The role of non-critical binding proteins in the sensitivity of acetylcholin-esterase from different species to diisopropyl fluorophosphate (DFP), [formula omitted

In vitro studies showed that organophosphate insecticides have different IC 50 values (i.e., concentration of inhibitor required to inhibit 50% enzyme activity) for acetylcholinesterases (AChE's) from different species. Since previous reports indicated that the binding of active cholinesterase inhibitors to non-critical binding proteins is an important mechanism of detoxification of organophosphate insecticides in vivo , we investigated the role of non-critical binding proteins in synaptosomal membrane preparations from monkey, rat and chicken brain tissues as a possible explanation of the differences in the IC 50's. The amount of non-critical binding proteins as well as critical binding sites, AChE, were determined by 3H-DFP binding in vitro . The affinity constants (Ka) and phosphorylation constants (Kp) of DFP for AChE's from these preparations were also determined by kinetic studies. In IC 50 studies, monkey brain AChE was 1.5X and 3.2X more sensitive to DFP inhibition than chicken and rat brain AChE. The observed differences in IC 50's cannot be explained on the basis of differences in the amount of non-critical binding proteins, since only small amount of non-critical binding proteins were found in these preparations. However, in the kinetic studies, monkey brain AChE had 3.7X and 2.1X higher affinity than chicken and rat brain AChE, respectively; and chicken brain AChE had about 2.7X faster rate of phosphorylation than the other two. It is therefore concluded that non-critical binding proteins are relatively unimportant in terms of affecting IC 50's. The differences observed in IC 50's are primarily due to different affinity and/or rate of phosphorylation of AChE active sites by DFP.

Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TW-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit.

Spectrin, fodrin, and TW-260/240 form a group of structurally and functionally similar but not identical high molecular weight actin-binding proteins from chicken erythrocytes, brain tissue, or intestinal epithelial brush borders. Immunological data and one-dimensional peptide maps of the separated subunits suggest that a common (Mr 240,000) and a variant (Mr 220,000, 235,000, or 260,000) subunit account for the three different heterodimers. These results are in line with the related but distinct morphology of the three proteins observed in micrographs of rotary-shadowed molecules and the finding that the common (Mr 240,000) subunit seems to account for the calcium-dependent calmodulin-binding activity displayed by the three proteins. The possible functions of spectrin-like molecules in nonerythroid cells are discussed.

Detection of beta-galactosyl(1 leads to 4)N-acetylglucosaminide alpha(2 leads to 3)-sialyltransferase activity in fetal calf liver and other tissues.

Using a micromethodology based on methylation, the specificity of sialic acid transfer to asialo-alpha 1-acid [3H]-glycoprotein in various tissues was studied. CMP-N-acetylneuraminyl: beta-galactosyl(1 leads to 4)N-acetylglucosaminide alpha(2 leads to 3)-sialytransferase activity (an activity which has not been demonstrated before) was detected in fetal calf liver, embryonic chicken brain, human placenta, and several other tissues. With the exception of the placenta all tissues investigated showed a considerable additional activity of CMP-N-acetylneuraminyl: beta-galactosyl(1 leads to 4)N-acetylglucosaminide alpha (2 leads to 6)-sialytransferase. Rat and porcine liver also contained the latter enzyme, but were essentially devoid of the alpha(2 leads to 3)-sialytransferase. Mixed enzyme experiments indicated that the alpha(2 leads to 3)-sialytransferase activity is due to a separate enzyme, which is clearly distinguishable from the CMP-N-acetylneuraminyl: beta-galactosyl(1 leads to 3)N-acetylgalactosaminide alpha(2 leads to 3)-sialytransferase of porcine liver and submaxillary gland.

Comparison of RF-induced calcium efflux from chick brain tissue at different frequencies: do the scaled power density windows align?

The recent suggestion that the pattern of positive and negative results of RF-induced calcium efflux from chick brain tissue, when appropriately scaled, matches at three frequencies is examined. Close scrutiny of this recently reported analysis by Joines and Blackman suggests that the uncertainties in the calculated scaling quantities are too large to permit meaningful conclusions.

Monoamine regulation of adenosine 3',5'-monophosphate in homogeneous neuronal cultures from chick brain hemispheres.

The effect of isoproterenol, norepinephrine, dopamine, and serotonin on the accumulation of adenosine 3',5'-monophosphate (cAMP) was studied in homogeneous neuronal cultures from 8-day chick embryo hemispheres. Among the catecholamines, isoproterenol had a more pronounced effect on the accumulation of cAMP. Norepinephrine and dopamine were considerably less potent and serotonin was ineffective. The response of neuronal cells to isoproterenol was inhibited by propranolol, suggesting that the cAMP increase was mediated by beta-adrenergic receptors. Maximally effective concentration of isoproterenol (10 microM) produced a 2.5-fold increase in cAMP content which is in contrast to the much greater cAMP response elicited by isoproterenol in chick brain tissue. These results suggest that in chick embryo hemispheres the nonneuronal cells are the major sites of the effect of beta-adrenergic agonists. The low responsiveness of the cAMP-generating system found in neuronal cultures is interpreted as reflecting either the number of beta-adrenergic receptors or the regulation of beta-adrenergic receptors and adenylate cyclase at the membrane level.

Calcium-ion efflux from brain tissue: power-density versus internal field-intensity dependencies at 50-MHz RF radiation.

In previous experiments changes were found in calcium-ion efflux from chick-brain tissue that had been exposed in vitro to 147-MHz radiation across a specific range of power densities when the field was amplitude modulated at 16 Hz. In the present study, 50-MHz radiation, similarly modulated as a sinusoid, was found to produce changes in calcium-ion efflux from chick brains exposed in vitro in a Crawford cell. Exposure conditions were optimized to broaden any power-density window and to enhance the opportunity to detect changes in the calcium-ion efflux. The results of a power-density series demonstrated two effective ranges: One spanning a range from 1.44 to 1.67 mW/cm2, and the other including 3.64 mW/cm2, which were bracketed by no-effect results at 0.72, 2.17, and 4.32 mW/cm2. peaks of positive findings are associated with near-identical rates of energy absorption: 1.4 microW/g at 147 MHz, and 1.3 microW/g at 50 MHz, which indicates that the enhanced-efflux phenomenon is more dependent on the intensity of fields in the brain than on the power density of incident radiation. In addition, the phenomenon appears to occur at multiples of some, as yet unknown, rate of radiofrequency (RF) energy absorption. Because of the extremely small increments of temperature associated with positive findings (less than 4 X 10(-4) degrees C), and the existence of more than one productive absorption rate, a solely thermal explanation appears extremely unlikely.

Utilization of Glucose in Slices of Chick Cerebral Hemispheres

Slices prepared from the 1- and 30-day-old chick cerebral hemispheres were incubated comparatively in a non-oxygenated and an oxygenated glucose-saline medium. The rate of swelling of the slices increased in the non-oxygenated medium, particularly for the 30-day-old chick preparations; the inulin space was the same for the two categories of slices and it increased in the oxygenated medium. An accumulation of glucose occurred mainly in the 1-day-old chick brain slices incubated in the oxygenated medium, and tissue lactate content increased in the non-oxygenated medium, particularly for the 30-day-old chick brain slices. Tissue pyruvate content was less dependent on the conditions of oxygenation, but tissue 2-ketoglutarate content increased in slices incubated in the oxygenated medium. The release of 14 CO2 by incubation of slices in the presence of (U-14 C)-glucose increased in the oxygenated medium mainly in the 30-day-old chick preparations, but (U-14 C)-glucose uptake seemed to be independent of conditions of oxygenation and age of the brain slices. Glutamate, 4-aminobutyrate, and glutamine levels were highest in the 30-day-old chick brain slices; asparate content was more stable in the 1-day-old chick brain preparations than in the 30-day-old chick ones; alanine and glycine levels were the highest in the 1-day-old chick brain tissues. The percentage of incorporation of 14C from (U-14C)-glucose was practically and respectively the same in the glucose, amino acid, and carboxylic acid fractions extracted from the 1- and 30-day-old chick brain slices incubated in the non-oxygenated medium, the radioactivity in the carboxylic acid fraction being the highest and that in the glucose fraction being the lowest. By incubation in the oxygenated medium, the 1-day-old chick brain slices showed high radioactivity in the glucose fraction and low radioactivity in the carboxylic acid fraction, and the reverse was true for the 30-day-old chick brain tissue; the labeling of the amino acid fraction was the same in the two categories of brain slices. All dicarboxylic amino acids have respectively the same specific radioactivity in the 1- and 30-day-old chick brain slices, and it was highest in the oxygenated medium. In all conditions of oxygenation the specific radioactivities in decreasing order were as follows: alanine, glutamate or aspartate, 4-aminobutyrate, glutamine, and glycine. The rate of utilization of glucose via the glycolytic and the respiratory pathways was less in the 1-day-old chick brain tissues than in the 30-day-old chick ones, although the metabolic pathways leading to the synthesis of the dicarboxylic amino acid seemed to be present in the two brain preparations.

Actin accumulation in developing chick brain and other tissues

Cytoplasmic actin isolated from chick brain tissue was identified by electron microscopy, myosin-binding ability, SDS and urea gel analysis, and tryptic peptide mapping. Tissue actin levels were measured from SDS-polyacrylamide gels of brain, heart, lens, liver, and skin tissue homogenates from 5- to 20-day chick embryos and from 17-week-old adults. Actin levels were highest in 11- to 13-day embryo brains. This time span is coincident with the most active period of neuron differentiation and axonal growth cone activity. Actin levels were generally higher in nonmuscle tissues of the embryo than of the adult, supporting the concept that actin is involved in cell motility during tissue morphogenesis. However, low levels of actin maintained in some adult nonmuscle tissues (brain, liver, and skin) suggest a role for cytoplasmic actin in physiological functions other than cell motility.

Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity.

Several rabbit antisera have been prepared against reduced and alkylated, electrophoretically purified tubulin isolated from chick brain. These antisera give a single precipitin line in Ouchterlony double diffusion plates when tested against partially purified tubulin, and label specifically microtubule- and tubulin-containing structures, such as mitotic spindles, cilia, and vinblastine-induced crystals, in a variety of cells. The same antisera also display the unique ability to stimulate the colchicine-binding activity of tubulin preparations from chick brain and Chinese hamster ovary tissue culture cells. This specific stimulation of colchicine binding activity is also obtained with the gamma globulin fractions purified by ammonium sulfate precipitation of these antisera.

Melatonin in the chicken brain: Its origin, diurnal variation, and regional distribution

What appears to be melatonin was demonstrated in chicken brain tissue. Brain melatonin activity was abolished by pinealectomy. There was a diurnal rhythm in brain melatonin, which, like the pineal melatonin rhythm, was high at night and low in the daytime. These findings suggest that the brain melatonin was of pineal origin. At midnight, the concentration of whole brain melatonin was five times higher than the serum melatonin level. Thus, brain tissue seems to concentrate melatonin. When analyzed by brain region, the highest level of melatonin was found in the hypothalamus, and the lowest level in the telencephalon, with the midbrain, cerebellum, and pons-medulla intermediate in concentration.

The Comparative Effect of Oral Ingestion of Methyl Mercury on Chicks and Rats

Two experiments with day-old chicks and one experiment with weanling rats were conducted to determine: (1) whether low levels of mercury found in some feedstuffs would result in hazardous accumulations of mercury in their tissues, and (2) the effects of feeding various dietary levels of methyl mercury to chicks and rats. Data show that feeding animal protein (fish meal) containing higher than normal levels of mercury did not result in the concentration of mercury in poultry tissues that exceeded FDA guideline levels. In fact, breast muscle concentration of mercury averaged only 0.1 p.p.m. even when the fish meal made up 17% of the diet. Significant amounts of mercury were deposited in the feathers of the test birds. At 16.9 p.p.m. dietary mercury as methyl mercuric chloride, all chicks succumbed to mercury toxicity by 17 days of age. Total mercury dose was 28.3 mg./kg. body weight. At this level of dietary mercury, brain, liver, and breast tissue of chicks at death contained 19, 47, and 15 p.p.m., respectively. Within limits, tissue concentrations of mercury were proportional to dietary levels. After 7 weeks of mercury exposure chicks retained an average of 63% of the mercury consumed. During a 21-day depletion period, chicks excreted an average of 0.66% of the total body burden of mercury per day. Thus biological half-lives can be calculated to average about 97 days for all dietary levels. Rats fed diets containing 16 p.p.m. mercury did not show outward signs of toxicity until the 6th week of treatment. After 12 weeks of mercury exposure, the total mercury dose was 84 mg./kg. body weight. None of the rats receiving lower levels of mercury showed symptoms of toxicity. Liver and brain mercury levels in rats fed 16 p.p.m. mercury averaged 26 and 12 p.p.m., respectively. Male rats generally had higher tissue levels of mercury than females. In general, rats tolerated the highest level of dietary methyl mercury for a longer period of time than chicks which consumed three times as much mercury and deposited less mercury in their tissues.

Transport systems for GABA and for other amino acids in incubated chick brain tissue during development

The transport of six amino acids (GABA(γ-aminobutyric acid), glycine, AIB (α-aminoisobutyric acid), leucine, d-glutamate, and lysine) was studied in brain slices from chick embryos and young chicks at different developmental stages. Initial accumulation rates increased significantly during embryonic development, and, with the exception of glycine, decreased somewhat after hatching. The greatest changes were observed with GABA. Steady state accumulation levels were reached more slowly and increased less than initial accumulation rates in more immature brains, suggesting that exit rates also increase during development. The influx of the six amino acids was analyzed kinetically: double-reciprocal plots of AIB, leucine, d-glutamate, and lysine influx gave straight lines at all the ages studied. For any of these amino acids, apparent K m values remained constant with age, but V values varied significantly, suggesting that the availability or the turnover of carriers, but not their affinity for the transported molecules, changes during development. Double-reciprocal plots of GABA and glycine influx gave two-limbed curves with embryonic brains (GABA) and also with young chick brain (glycine), which seems to indicate that at least two modes of mediated entry exist for these amino acids. With GABA one of the two transport systems tends to disappear, or to become undetectable, with increasing age. Substrate specificity studies of GABA influx showed that only amino acids having the cationic group in beta or gamma position are effective inhibitors of GABA transport. The developmental pattern of the inhibitions obtained at selected GABA concentrations suggests that a high affinity and low capacity transport system for GABA tends to disappear during development. Changes in the pattern of the sodium dependence of GABA influx also indicate the existence of more than one transport system at early developmental stages. When the substrate specificity and the sodium-dependence of AIB influx were studied, similar inhibitory effects were obtained at different AIB concentrations, confirming that this amino acid is transported by only one transport system. However, a progressive increase in the overlapping between AIB and leucine transport systems during development was demonstrated by the progressively increasing inhibition of AIB influx by leucine.