Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A₁₂ collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A₁₂ activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G₄ species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G₂ and G₁ forms are present in comparable concentrations in both tissues. Besides, G₄ is the predominant globular form in the chick optic lobe after hatching, G₂ and G₁ being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered.The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.     

Benzodiazepine receptor sites in the chick optic lobe: Development and pharmacological characterization

To investigate the, interaction between -aminobutyric acid (GABA) and benzodiazepine (BZD) receptor sites during development, the time-course of appearance of flunitrazepam (FNZ) binding sites and their pharmacological characterization were studied in developing chick optic lobe. At the earliest stage examined, embryonic day (Ed) 12, the receptor density was 30.9 % (0.05±0.01 pmol/mg protein) of that found in the chick optic lobes of adult chicks. The adult value was achieved on Ed 16 (0.16±0.01 pmol/mg protein). After this stage there was a sharp and transient increase in specific [³H]FNZ binding of about two-fold reaching a maximal value between hatching and the postnatal day (pnd) 2 (0.33±0.01 pmol/mg protein). Scatchard analysis at different stages of development revealed the presence of a single population of specific FNZ binding sites. The increase in [³H]FNZ binding during development was due to a large number of binding sites while their affinity remained unchanged. Competition experiments in the chick optic lobe revealed that the order of potency for displacement of specific [³H]FNZ binding paralleled the pharmacological potency of the BZDs tested. The IC₅₀ s for clonazepam, flunitrazepam, Ro 15-1788 and chlordiazepoxide were 3.02, 4.30, 0.32, and 4778.64 nM respectively. Ro 5-4864, a potent inhibitor of BZD binding to peripheral tissues, had no effect on specific [³H]FNZ binding indicating that only central BZD binding sites are present in the chick optic lobe. The peak of maximal expression of BZD receptor sites precedes in 5–6 days the peak of GABA receptor sites indicating a precocious development of BZD receptor sites. The different appearance of both peaks may represent important events during development probably related to synaptogenesis.     

Developmental Pattern of Plasminogen Activator Activity in Chick Brain Hemispheres

Plasminogen activators play key roles in several developmental events. In previous works we demonstrated the existence of typical developmental patterns of protease activity in the chick optic lobe and cerebellum. The aim of this work is to study the temporal pattern of development of plasminogen activator activity in the brain hemispheres. Plasminogen activator activity was assayed in soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions by using a radial fibrinolytic assay. Employing different inhibitors and anti-plasminogen activators antibodies we showed that developing brain hemispheres express only one type of enzyme which corresponds to the urokinase-type. Other results indicate that the protease activity displays a temporal pattern which completely differs from those of general parameters of development. This suggests that the plasminogen activator activity is developmentally regulated and could display specific functions during particular stages of development.     

Glutamate decarboxylase activity in chick brain and retina

We have studied the effect of Triton-X-100 on glutamate decarboxylase (GAD) activity in brain and retina from chick embryos of 12 and 16 days' incubation and from chicks 4–6 weeks old. GAD activity was measured in five different homogenization media. Triton-X-100 inhibited the enzyme by about 60% in both brain and retina of 12-day embryos and by about 50% in 16-day embryos, independently of the homogenization medium. In chicks only about 20% inhibition by the detergent was observed in brain whereas no effect was found in retina. These results indicate that the evaluation of the experimental conditions of enzyme assays at different ages is essential for developmental studies of GAD activity in nervous tissue.     

Light and dark adaptation influences GABA receptor sites in the chick retina

The aim of the present study was to investigate the effect of environmental conditions such as light-and-dark-adaptation on the plasticity of GABA receptor sites in the chick retina. In chicks exposed to light for 5 hr (light-adapted), specific [³H]GABA binding was increased by 35% in comparison to the binding found in chicks maintained in darkness (dark-adapted). Conversely, in the retina of chicks exposed to darkness for 5 hr, specific [³H]GABA binding was decreased by 28% with respect to that found in chicks kept in the light. Scatchard analysis of the binding data revealed that the affinity of GABA for its receptor binding site was higher in the retinas of light-adapted chicks than in those of dark-adapted chicks (K _d values of 19.20±1.23 and 27.20±1.47 nM, respectively). On the contrary, the maximal number of binding sites (B_max) remained unchanged in light- and dark-adapted chicks (5.2±0.10 and 5.3±0.15 pmol/mg protein, respectively). These results suggest the involvement of GABA receptors in the regulation of visual function.Special Issue dedicated to Prof. Eduardo DeRobertis     

Comparative study of miscellaneous properties of cysteine sulfinate transaminase and glutamate oxaloacetate transminase in chick retina homogenate

The activity, properties, and developmental pattern of cysteine sulfinate transaminase (CSA-T) were studied in chick retina and compared with the activity, properties, and developmental pattern of glutamate oxaloacetate transaminase (GOT). Their optimum pH is identical whereas the effect of pyridoxal phosphate seems to be different. Developmental patterns are also different. TheK _m andV _m of CSA-T and GOT were determined in chick retina homogenate. These results suggest that two different enzymes are responsible for the transamination of cysteine sulfinate (CSA) and aspartate.     

Cysteine sulphinate decarboxylase in chick and rat retina during development

The activity, properties and developmental pattern of cysteine sulphinate decarboxylase (CSD) were studied in chick and rat retina. Retinal CSD shows properties similar to those of the enzyme in brain with respect to optimum pH, saturating substrate concentrations and stimulation by pyridoxal phosphate, CSD activity increased 3-fold from the 10th day of embryogenesis to hatching in chicks and in postnatal development in rats. The developmental pattern of CSD activity in both species is coincident with the functional maturation of visual function.     

3β-OH-5β-Pregnan-20-One Enhances [3H]Gaba Binding in Developing Chick Optic Lobe

Abstract

The neurosteroids are synthesized in the CNS and act mainly through allosteric modulation of the GABA_A receptor. Structure-activity relationship studies in mammalian CNS have shown that a 3α-hydroxyl group and a 5α-reduced A-ring are striking features for their biological activity, while the 3β,5β structures as in 3β,5β-P are completely inactive. In this work we report the enhancing activity of epipregnanolone on [³H]GABA binding to its receptor sites in the chick optic lobe. Concentration-effect curves for this neurosteroid showed a concentration-dependent activity with different potencies at the three developmental stages studied, the hatching stage being the most sensitive to the steroid stimulatory effect. The displacement of a potent 3α,5α steroid by epipregnanolone indicated that this steroid behaved as a partial agonist of the steroid recognition site. Considering the developmental profile for steroidogenesis in avian tissues and the biological relevance of 5β-reduced steroids in early development, we propose that 3 or its 3α-epimer, pregnanolone, instead of the potent 3α,5α neurosteroids, modulates GABA_A receptors in the chick optic lobe during development.     

Carnitine,carnitine acetyltransferase,and glutathione in Alzheimer brain

Glutathione and total carnitine (i.e., free carnitine plus acid-soluble carnitine esters) were measured in an affected (superior frontal gyrus; SFG) and unaffected (cerebellum: CBL) region of Alzheimer disease (AD) and control brains. Average glutathione content in AD SFG (n=13) and AD CBL (n=7) (7.9±2.1 and 11.9±4.0 nmol/mg protein, respectively (mean ±S.D.)) was similar to that in control SFG (n=13) and CBL (n=6) (7.7±2.0 and 11.6±2.6 nmol/mg protein, respectively). However, glutathione increased significantly with age in AD brain (p=0.003) but not in control brain. Average total carnitine in AD SFG (84±47 pmol/mg protein; n=10) and AD CBL (108±86 pmol/mg protein; n=7) was not significantly different from that in the corresponding regions of control brain (148±97 (n=10) and 144±107 (n=6) pmol/mg protein, respectively). However, a significant decline of total carnitine with age in both regions was noted for AD brain, but not for control brain. Carnitine acetyltransferase activity in the AD SFG (n=13) was not significantly different from that of control SFG (n=13) (1.83±1.05 and 2.04±0.82 nmol/min/mg protein, respectively). However, carnitine acetyltransferase activity of AD CBL (n=7) was significantly lower than that of control CBL (n=6) (1.33±0.88 versus 2.26±0.66 nmol/min/mg protein; p=0.05).     

Experssion of α-Bungarotoxin Receptro Subtypes in Chick Central Nervous System During Development

Abstract

Chick central nervous system (CNS) expresses a-bungarotoxin (aBgtx) receptors. We have recently reported the purification and characterization of two aBgtx receptor subtypes, a7 and a7-a8 from chick optic lobe (COL). In order to study whether other aBgtx receptor subtypes are present in other areas of the chick CNS, as well as their developmental expression, we used anti-a7 and anti-a8 subunitspecific antibodies to study aBgtx receptors at different developmental stages in COL, brain and retina. We found that only the a7 and a7-a8 subtypes are present at all developmental stages in chick COL and brain, where they represent 90% of all the aBgtx receptors at embryonic day 19 and 1 day post hatching (Dl). In chick retina, an a8 subtype representing 50% of all aBgtx receptors at D1 is present in addition to the a7 and a7-a8 subtypes, and the expression of this a8 subtype increases during neurodevelopment.     

Divergent Regulation of Muscarinic Binding Sites and Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain

Summary The expression of muscarinic acetylcholine binding sites and of cholinesterases was studied in extracts prepared from discrete regions of the human fetal brain, between the gestational ages of 14 and 24 weeks. The specific binding of [³H]N-methyl-4-piperidyl benzilate ([⁴H]-4NMPB) to muscarinic binding sites ranged between 0.05 and 1.30 pmol/mg protein in the different brain regions, withK _d values of 1.2 ± 0.2 nM. Binding of the cholinergic agonist oxotremorine fitted, in most of the brain regions examined, with a two-site model for the muscarinic binding sites. The density of muscarinic binding sites increased with development in most regions, with different rates and onset times. It was higher by about sixfold in some areas destined to become cholinergic, such as the cortex and midbrain, than in noncholinergic areas such as the cerebellum. In other areas destined to become cholinergic, such as the hippocampus and the caudate putamen, the receptor density remained low. Average density values increased from 0.1 ± 0.1 at 14 weeks up to 0.7 ± 0.4 pmol/mg protein at 24 weeks.The variability in the specific activities of cholinesterase was relatively low, and extracts from different brain regions hydrolyzed from 5 to 30 nmol of [³H]acetylcholine/min/mg protein. These were mostly true acetylcholinesterase (EC 3.1.1.7) activities, inhibited by 10^–5 M BW284C51, with minor pseudocholinesterase (EC 3.1.1.8) activities, inhibited by 10^–5 M iso-OMPA. The enzyme from different brain regions and developmental stages displayed similarK _m values toward [³H]acetylcholine (ca. 4 × 10^–4 M ^–1). The ontogenetic changes in cholinesterase specific activities had no unifying pattern and/or relationship to the cholinergic nature of the various brain areas. In most of the brain regions, the arbitrary ratio between the specific activity of cholinesterase and the density of muscarinic binding sites decreased with development, with average values and variability ranges of 83 ± 50 and 19 ± 19 at 14 and 24 weeks, respectively. Our findings suggest divergent regulation for cholinergic binding sites and cholinesterase in the fetal human brain and imply that the expression of muscarinic receptors is related to the development of cholinergic transmission, while acetylcholinesterase is also involved in other functions in the fetal human brain.I.B. took part in this work as partial fulfillment of the requirements of the Sackler Faculty of Medicine for an M.D. degree.     

Calmodulin-dependent protein phosphatase: a developmental study

Calmodulin-dependent protein phosphatase, one of the major calmodulin-binding proteins in bovine brain, dephosphorylates casein with a specific activity of 15 nmol mg-1 min-1 at 30 degrees C. The stimulation of phosphatase activity by calmodulin is reversed by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or trifluoperazine, a calmodulin antagonist. Antibodies raised in rabbit against the phosphatase inhibit the enzyme activity. The levels of the protein in brain extracts from various animals, determined by a radioimmunoassay, range from 20 micrograms/g of tissue in chick and fish brains to 143 micrograms in rat cerebrum. The ontogeny of the phosphatase was studied in nervous tissues from rat and chick, animals in which synaptogenesis takes place at different times during their development. The levels of the protein increased significantly in rat cerebrum and cerebellum and in chick brain and retina during the periods corresponding to major synapse formation. In rat cerebrum, the enzyme appeared to be equally distributed between the cytosol and the particulate fraction; the level in both compartments increased during the major period of synapse formation. Thus, the development of calmodulin-dependent protein phosphatase closely parallels synaptogenesis, implicating a role in some synaptic function.     

Ontogenesis of Muscarinic Receptors and Acetylcholinesterase Activity in Various Areas of Chick Brain

Abstract: Muscarinic receptors, labeled with [³H]quinuclidinyl benzylate (³H]QNB), and acetylcholinesterase activity were studied in five areas of the developing chick brain: (1) hyperstriatum and neostriatum , (2) paleostriatum, (3) optic lobes, (4) mesodiencephalon and (5) cerebellum. The protein content of these areas, expressed as mg/g tissue and total protein, was determined between day -10 and adulthood. Differences in both determinations were observed among the areas. The binding of [³H]QNB was expressed as density (fmol/mg protein) and total number of receptors (fmol/total protein) in the area. Considerable variations were observed among the areas. The cerebellum showed the lowest receptor density and a large decrease in density and total number of receptors in the adult, which may reflect a change in neuronal population. Acetylcholinesterase, in certain areas, accompanied the changes in receptor concentration, but the timing and rate of increase had special features in each case. The most striking one was the cerebellum, in which the enzyme increased steadily postnatally, while the muscarinic receptors dropped to very low values.     

Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

Developmental Changes in β-Citryl-L-G1utamate Concentration and Its Synthetic and Hydrolytic Activities in Neuronal Cells Cultured from Chick Embryo Optic Lobes

Developmental changes in the concentration of beta-citryl-L-glutamate(beta-CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by beta-CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of beta-CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into beta-CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of beta-CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.     

The development of superficial infraslow potential oscillations of the brain in chick embryos.

The development of superficial infraslow potential oscillations (ISPO) of brain hemispheres, cerebellum and optic lobes was studied in chick embryos between day 9 and 21 of incubation. The ISPO were firstly registered in brain hemispheres at day 10 of incubation, i.e. 5 days before the onset of spontaneous EEG activity. The ISPO in 10-day-old embryos had an average frequency of 9.9 c/min and an amplitude of 0.14 mV. During further development till hatching the frequency decreased to 7.5 c/min and the amplitude increased to 1.39 mV. Similar ISPO with the same developmental trend were also registered from the surface of the cerebellum and optic lobes. Superficial ISPO were not synchronized either between both hemispheres or between different fields of the same hemisphere.     

Partial characterization of histidine decarboxylase in hamster and rat brain employing a new method

Histidine decarboxylase activity in hamster and rat brains were studied using a newly developed sensitive, direct radioenzymatic microassay. For our assay conditions, we determined aK _m forl-histidine of 320 M and aV _max for histidine decarboxylase of 110 pmol histamine/hr/mg protein in rat hypothalamus. The regional distributions of both histidine decarboxylase and histamine levels were similar in the hamster and rat with the most activity in hypothalamus. Most of the histidine decarboxylase activity in rat hypothalamus was in the cytosol fraction. The developmental pattern of histidine decarboxylase in the fetal rat did not reveal a prenatal spike in activity. Histidine decarboxylase activity in rat brain reached adult levels by four weeks. Alpha-fluoromethylhistidine inhibited histidine decarboxylase activity almost totally in vitro at 10 M and about 80% in vivo after six days of infusion (100mg/kg/day) in all brain regions except the cerebellum. Likewise, histamine levels were depleted about 75% in all brain regions except the cerebellum.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Myocardial Ca-sequestration failure and compensatory increase in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using real-time ratiometric indo-1 spectrofluorometry

A novel, simple, rapid and reproducible microassay is used for kinetic analysis of Ca-sequestration by homogenates of myocardium of turkeys with furazolidone-induced congestive cardiomyopathy. The assay monitors Ca in real-time using dual-emission ratiometric spectrofluorometry and the Ca-indicator dye indo-1. Using this assay and isolated SR studies we make several novel findings regarding the mechanism of SR failure in furazolidone cardiomyopathy.Qualitative differences in Ca-sequestration were not detected between groups. However, compared to controls the furazolidone treatment resulted in: 1) 50% depression in maximal activities (1.54 ± 0.36 vs 0.73 ± 0.12 µM/sec); 2) 2-fold increases in post-sequestration concentrations of ionized Ca(79 ± 23 vs 141 ±13 nmol Ca/L homogenate); 3) 2-fold increases in Ca half-life (415 vs 790 msec); and 4) 25% increased passive Ca-binding capacity of homogenates. The Ca-ATPase specific activity of isolated sarcoplasmic reticulum was 60% increased in congestive cardiomyopathy (543 ± 140 vs 873 ± 108 nmol ATP hydrolyzed/min/mg membrane protein) although membrane yield was 20% decreased (0.79 ± 0.09 vs 0.63 ± 0.03 mg/g heart). The increased ATPase and decreased Ca-uptake activities in combination with the occurrence of 36% cardiac hypertrophy and 19% decreased body weights resulted in estimates of the relative energy cost to the animal for myocardial Ca transport being 5.5-fold increased with cardiomyopathy (20.5 vs 111 nmol ATP hydrolyzed per µM decrease of sarcoplasmic free Ca/kg body weight).These data indicate that congestive cardiomyopathy is associated with markedly increased permeability of sarcoplasmic reticulum to Ca and compensatorily increased Ca-ATPase activity. Accelerated energy consumption due to the increased energy cost of Ca transport and increased time of myocyte activation are predicted to predispose the myocardium to fatigue and irreversible failure.