Characterization of experimental phenylketonuria. Augmentation of hyperphenylalaninemia with alpha-methylphenylalanine and p-chlorophenylalanine.

Phenylalanine in conjunction with p-chlorophenylalanine or alpha-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterious effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing alpha-methylphenylalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to experimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, gamma-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in the brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age exhibited a long-term learning impairment. These observations are consistent with many aspects of the clinical picture of untreated phenylketonuric patients, and suggest that this animal model will be beneficial in studying the disease.

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture.

Oligodendrocytes were isolated from adult pig brain and cultivated for 18-24 days. [(14)C]acetate, [(3)H]galactose or [(35)S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [(14)C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [(14)C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [(14)C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [(3)H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [(35)S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.

Altered precursor availability and metabolism of monoamines in the brain caused by the injection of physiologic saline to suckling rats: On the reliability of saline "controls" in neurochemical studies.

The effect of subcutaneous injections of saline (0.9% NaCl, 10-40 ?l/g b. wt) to 5- and 20-day old rats on the concentrations of tyrosine (Tyr) and tryptophan (Trp) in the serum and the brain and on the levels of biogenic amines and their metabolites in the developing brain at 6 h p.i. is described. At day 5 the concentration of Tyr in the blood was decreased (dose-dependent), but the brain concentrations of Tyr and of its amine-metabolites, dopamine (DA), norepinephrine (NE), homovanillic acid (HVA) and dihydroxyphenylacetate (DOPAC) were unaffected. In contrast, in the 20-day old rat, serum Tyr was unaffected by the saline injections, but the Tyr concentration in the brain decreased markedly at the highest saline dose. The concentrations of NE (only at maximum dose) and of DA (independent on the amount of saline injected) were elevated in the brains of saline injected 20-day old rats. The concentrations of Trp and indoles were more affected at day 5 than at day 20: slightly decreased concentration of Trp in the serum but markedly increased concentrations of brain Trp (only at maximum dose), elevated serotonin (5-HT, independent on the amount of saline injected) and 5-hydroxyindoleacetic acid (5-HIAA, at maximum dose) in the brain. If the maximum dose of 40 ?l/g body weight was injected to suckling rats repeatedly during the whole suckling period (in 12 h intervals), some effects caused by one single injection of 40 ?l/g disappeared (Tyr-depletion in blood or brain, increase in brain NE, DA and Trp), but other additional effects appeared (decreased DA and increased DOPAC, decreased 5-HT and 5-HIAA). The results show that saline injections do cause characteristic, age-dependent alterations of precursor availability as well as of the rate of synthesis and degradation of catecholamine and 5-HT. Repeated treatments have different effects than one single treatment on the precursor availability and the metabolism of monoamines. These alterations must be taken into account if the effects of certain "specific" treatments are compared and discussed in relation to saline "controls".

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker.

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient. The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.

Nervous system myelin in the electric ray, Torpedo marmorata: Morphological characterization of the membrane and biochemical analysis of its protein components.

In Torpedo, PNS as well as CNS myelines are characterized by clearly separated double intraperiod lines. CNS myelin of Torpedo contains two glycosylated hydrophobic proteins labelled T1 (25,800 Da1) and T2 (29,700 Da1), and two basic proteins BP1 and BP2, migrating like mammalian large basic protein (BP2) and pre-small basic protein (BP1) (Barbarese et al., 1977). PNS myelin of Torpedo carries only BP1 and is characterized by a closely spaced doublet of the glycosylated hydrophobic proteins Con A+ (29,700 Da1) and Con A? (31,000 Da1); the latter does not bind Concanavalin A. These glycosylated proteins (T1, T2, Con A+, Con A?) contain mannose, N-acetylglucosamine and galactose, but lack fucose and sialic acids. They have isoleucine at their amino terminus. They bind anti-rat PNS myelin P(0) antibodies but do not react with anti-rat CNS myelin PLP antibodies. Limited proteolyses of isolated proteins suggest sequence homologies between T1 and T2, and possibly between Con A+ and Con A?. The two basic proteins BP1 and BP2 bind antibodies directed against human myelin basic protein. All Torpedo myelin proteins electrofocus in pH regions characteristic of their mammalian counterparts.

Characterization of a myelin-related fraction (SN 4) isolated from rat forebrain at two developmental stages.

A myelin-related fraction (SN 4) was isolated from forebrain of 17- and 40-day-old rats. Fraction SN 4 was obtained as a supernatant in a slow speed differential centrifugation of a myelin fraction. In contrast to multilamellar myelin fraction, SN 4 consisted of small vesicular profiles of a mixture of single membranes and some triple-layered structures. All typical myelin components were found in the SN 4 fraction from adult rat brain but their relative proportion was different from that of myelin: Wolfgram protein, myelin glycoproteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase were increased, while basic proteins and proteolipid protein were decreased significantly. In contrast, the lipid composition appeared very similar to the one found in myelin. SN 4 from 17-day-old rat brains was essentially similar to that from adults, except that the major myelin glycoprotein was not enriched in comparison to myelin. Developmental changes found in myelin were also present in the SN 4 fraction. The specific radioactivity of the fucose-labeled major myelin glycoprotein was similar in SN 4 and in myelin. The particular composition of fraction SN 4 suggests that this material is not significantly contaminated by non-myelin-related membranes but rather supports the hypothesis that it could be enriched in a membrane representing a zone of transition during the formation of myelin and which is subjected to a remodelling of its protein components.

Dopamine metabolism in characterised neurones of Planorbis corneus.

A sensitive chromatographic procedure was used to study the metabolism of [14C]tyrosine, [3H]DOPA and [3H]dopamine in 3 defined cell-types situated in the nervous system of Planorbis corneus. One of the cell-types contains dopamine (GDC), the other serotonin (GSC) and the other neither amine (GC). The GDCs metabolise [14C]tyrosine to form DOPA and dopamine while the other two cells lack this ability. In contrast, the GDCs and the GSC, but not the GCs, metabolise [3H]DOPA to form dopamine. In addition the GDCs incorporate radioactivity from [3H]DOPA into DOPAC, homovanillic acid and methoxytyramine. After incubation of cells in [3H]dopamine, only the GDCs metabolise it to form DOPAC, homovanillic acid and methoxytyramine. In no instance did the GDCs form significant amounts of noradrenaline from the incorporated radioactive substances. These results, together with data on the amine histochemistry of the individual cell-types following pretretment of animals with drugs known to affect specific enzymes in the synthesis of amine transmitter substances, clearly demonstrate that the GDCs alone have the enzymes requisite for the biosynthesis and catabolism of dopamine, but not noradrenaline.

Myelin protein composition in the rat spinal cord in culture and in vivo: a developmental comparison.

Biochemical characterization of the development of myelin in vitro was extended to an analysis of myelin protein composition in cultures of explanted foetal rat spinal cord. Myelin fractions were isolated from pooled explants after 12-30 days in vitro and, for comparison, from the spinal cords of rats of equivalent developmental ages. Electron microscopic examination of the culture myelin fractions revealed the presence of multilamellar myelin fragments and some single membranes. All fractions were analyzed using a micro-polyacrylamide gel electrophoresis technique. Qualitatively similar protein profiles were observed for myelin isolated from either cultures or from spinal cords. Fractions from cultures contained a greater proportion of high molecular weight proteins than those from spinal cords, although with respect to the 'major' myelin proteins, a quantitatively similar developmental pattern was observed both in vivo and in vitro.

The accumulation of DL-glutamate by the central nervous system of the snail Helix pomatia.

Isolated snail ganglia are capable of maintaining their free amino acid levels steady for the first 60 min of incubation in physiological saline. Within this time the ganglia also possess an uptake mechanism for DL-glutamate which can be divided into sodium-sensitive and -insensitive components. The accumulation of DL-glutamate showed saturation kinetics typical of a carrier-mediated process. The Vmax value for the uptake is 1.5 x 10(-8) mole/g/min and the Km value 1.1 x 10(-4) M. The amino acid accumulation is quite specific towards L-dicarboxylic acids and insensitive to a number of metabolic inhibitors. It is unlikely to be due to a homoexchange phenomenon because the ganglia are capable of achieving a net uptake of glutamate and the efflux of DL-[3H]glutamate is not increased by the addition of non-radioactive L-glutamate to the incubation medium.

Oligodendrocytes from postnatal cat brain in cell culture. I. Regeneration and maintenance.

Oligodendrocytes isolated in bulk from white matter of cat brain (8-12 weeks of age) employing a Percoll gradient as the final purification step, were cultured and maintained for more than 10 weeks. Different parameters, e.g. coating material and the age of the animals appeared to have some influence on attachment rate and survival of the cells. Oligodendrocytes from older animals, or oligodendrocytes seeded into poly-L-lysine coated culture dishes revealed a marked tendency to form aggregates. Of the dispersed cells, 80-99% can be classified as oligodendrocytes by transmission electron microscopy (TEM) and immunocytochemical markers. Aggregates which were re-seeded consisted of more than 90% oligodendrocytes. About one day after attachment to the supporting layer the cells start to regenerate their processes which sometimes broaden at their ends into shovel-like, membranous extensions.

Neurochemical and morphological studies of bulk isolated rat brain cells. II. Preparation of viable cerebral neurons which retain synaptic complexes.

The bulk isolation from rat cerebral cortex of viable neurons retaining synaptic complexes is described. The basis of this procedure is to dissociate the neurons in situ from the surrounding glial cells. The glial structures that are normally adjacent to the neuronal cell body and to the proximal parts of the neuronal processes are largely destroyed by perfusion of the brain under special conditions. The most important of these conditions was found to be a hyperosmolar concentration of hexoses in the perfusion medium. In addition, the presence of collagenase and hyaluronidase in the perfusion medium and specific perfusate flow characteristics were required to produce the structural changes throughout the brain tissue. When the perfused brain was further dissociated into a cell suspension by mincing and sieving, isolated neurons were obtained, the majority of which retained the proximal parts of their processes. A novel feature of these neurons was the retention of synaptic boutons on the plasma membrane. Presynaptic terminals with mitochondria and vesicles as well as pre- and postsynaptic membranes and densities were observed on the isolated neurons. The neurons were fractionated to 90--95% purity using discontinuous Ficoll density gradient centrifugation with a liquid fluorocarbon as cushion. Highly purified, viable cerebral neurons retaining synaptic complexes are thus available in bulk for neurobiological studies.

The binding of tetanus toxin to retinal cells.

An indirect immunofluorescence assay demonstrated intense binding of tetanus toxin to a minority of cells cultured from neonatal rat retina. These labelled cells were typically spheroid cells bearing long processes. Flat cells and smaller spheroid cells without long processes were essentially unlabelled. A similar assay performed on frozen sections of rat retina showed that tetanus toxin bound intensely to the inner plexiform layer, moderately to the outer plexiform and inner nuclear layers, and only very slightly to the outer nuclear layer. The localization of tetanus toxin bound to frozen sections was confirmed by autoradiography of [125I]tetanus toxin binding. In contrast, the monoclonal antibody A4 bound to all layers of the retina (except that of the outer segments). In cell cultures the monoclonal antibody Ran-2 bound to the flat cells but to neither class of spheroid cells. These data imply that tetanus toxin binds more intensely to retinal neurones than to photoreceptors and other retinal cell types. By assisting identification of cells dissociated from retina, assays of tetanus toxin binding should prove useful in the isolation of specific retinal cell-types.

Central nervous system myelin proteins and glycoproteins in vertebrates: a phylogenetic study.

CNS myelin was isolated by a conventional method from a wide range of vertebrate classes and analyzed by SDS-PAGE for proteins (Coomassie blue) and glycoproteins (concanavalin A (Con-A)-peroxidase). Mammalian, avian and reptilian myelin shared similar protein patterns (basic protein, BP; intermediate protein, DM-20; proteolipid protein, PLP; Wolfgram protein, W). Amphibians lacked DM-20 but were characterized by specific activities of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) higher than those of the other classes examined. The Con A-binding profiles were similar in the high molecular weight (HMW) regions of the classes listed above, while the typical myelin proteins in the low molecular weight (LMW) regions were devoid of Con A-binding properties. In teleost myelin a putative BP band ran well ahead of rat small basic protein (SBP), whereas the region corresponding to rat PLP was covered by several closely spaced bands, most of which bound Con A. In elasmobranch myelin, apart from bands corresponding to BP, Con A-binding glycoproteins were detected migrating in the region of rat DM-20 and PLP as well as with mammalian PNS P0 protein. Cyclostomates yielded only very small amounts of material in the myelin preparation and displayed undifferentiated Coomassie blue- and Con A-binding in the HMW region, while typical LMW myelin proteins were absent. These results demonstrate that CNS myelin from bony and cartilaginous fishes is characterized by containing several major Con A-binding proteins of low molecular weight. This is in striking contrast to myelin from phylogenetically higher classes.

Regional protein patterns of the embryonic chick brain: developmental shifts and the effects of light stimulation.

The developmental changes in the regional protein patterns of the embryonic brain of chick were analysed by micro-two-dimensional polyacrylamide gel electrophoresis stained by a highly sensitive procedure with Coomassie Brilliant Blue G-250 that completely avoids background staining. More than 400 protein spots were resolved in a computerized plot. Many proteins of the various embryonic brain regions varied with age. At least six of these proteins changed remarkably during development. One of them (Mr 14K) was prominent at 6 days and disappeared after 12 days of incubation. One acidic protein (Mr 30K) appeared at 12 days together with two acidic protein spots near tubulin and increased with development. A group of more basic proteins (Mr 94K) was prominent at 6 day incubation and decreased during development. One more basic protein (Mr 20K) was first observed at 18 days of incubation in the metencephalon. It was identified as myelin basic protein, and strikingly increased in all brain regions until hatching. A group of basic proteins with high molecular weight (Mr 94K) appeared at day 12 of incubation, and increased remarkably before hatching. They were identified to be collagen associated with brain capillary vessels. Stimulation by intermittent light in ovo from day 10 to day 16 caused a retarded appearance of these collagen proteins, and an increased formation of tubulin in the optic lobe of the developing chick embryo.

Regulation of the amino acid availability in the developing brain. No physiological significance of amino acid competition in experimental hyperphenylalaninemia.

Chronic experimental hyperphenylalaninemia in suckling rats causes a depletion of amino acids in the blood and in the brain, and an accumulation of amino acids in the peripheral tissues. The amino acid depletion in the blood is greater than that in the brain. The amino acid accumulating potency of all body tissues is increased by the excess of phenylalanine, most pronounced in the gut, least pronounced in the brain. All body tissues compete for the amino acids circulating in the blood. This competition is enhanced in hyperphenylalaninemia. The brain is at a disadvantage in the competition of the various body tissues for the amino acids available from the common pool. Brain tissue is increasingly depleted of amino acids as the accumulation of amino acids in the peripheral tissues is stimulated in hyperphenylalaninemia. The depletion of amino acids in the blood and the simultaneous rise of the free amino acid concentrations in the various developing tissues indicates tissue-specific shifts in the balance between protein synthesis and protein degradation in hyperphenylalaninemia. There is no indication that amino acid competition at the blood-brain barrier contributes importantly to the depletion of amino acids in the brain tissue in hyperphenylalaninemic rats. Instead, brain amino acid pools under in vivo steady-state conditions appear to be primarily regulated by the rate of amino acid utilization by the peripheral tissues.

Myelination in rabbit optic nerves is accelerated by artificial eye opening.

Artificial opening of the eyes of young rabbits on the 5th postnatal day led to accelerated myelination: the myelin-specific basic and proteolipid proteins nearly doubled between the 7th and the 10th postnatal days when compared to controls; 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity also increased by about 60%. Conversely, lowered AChE activities presumably reflected elevated myelin/axolemma ratios. Myelination in treated animals normalized during later ontogenetic stages (greater than 20th postnatal day).

The lipid composition of light and heavy myelin subfractions isolated from rabbit sciatic nerve.

Centrifugation of adult rabbit sciatic nerve homogenates on continuous sucrose density gradients yields three distinct maxima at 0.12, 0.33 and 0.57 M sucrose (labeled A, B and C), respectively. Fraction A is absent in homogenates of immature rabbit sciatic nerve and the material isolated from adult animals is found to contain large amounts of triglycerides. It is proposed that fraction A results from the presence of adipose tissue adhering to the adult nerve preparations and should not be regarded as a true myelin subfraction. The concentration of the major membrane lipid classes (phospholipid, cholesterol and galactosyl ceramide) decreases from the light to the heavy side of the gradient. The ethanolamine phosphatide to sphingomyelin ratio increases from 1.06 to 1.42 between fractions B and C, due to an increase in the phosphatidal ethanolamine content at the expense of sphingomyelin.

Myelin composition in the central nervous system of mutant Han-Wistar rats.

Characterization of a Han-Wistar mutation (which is expressed as progressive spastic paresis in homozygotes) was extended to an analysis of myelin isolated from the brain, cerebellum and spinal cord of affected and littermate control rats. In all regions, the tissue content and protein composition of myelin from both groups of animals were similar, indicating that the synthesis of abnormal myelin is not the primary effect of this mutation.

Carbonic anhydrase activity in myelin fractions from rat optic nerves.

The carbonic anhydrase activity of myelin fractions isolated from the optic nerves of adult and immature (20-day-old) rats was examined. The specific activity in both total homogenate and myelin fractions was about 2-fold higher in adult than in immature animals and at both ages, the activity in the homogenate was higher than in myelin. After subfractionation by zonal gradient centrifugation, it was shown that carbonic anhydrase activity was greatest in the heaviest myelin particles at both ages. These data are consistent with the hypothesis that a small proportion of the total enzyme activity is localised in myelin.

Localization of tyrosine-hydroxylase immunoreactive cells in rabbit retinal cultures.

Tyrosine-hydroxylase immunoreactivity is demonstrable by the indirect immunofluorescence technique in a small sub-population of retinal neurones cultured from 2-day-old rabbits. Staining was only weak, restricted to a few neurones, and could not be observed in cultures of less than 10 days. Autoradiographical analysis of [3H]dopamine uptake by retinal cultures strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity have the capacity to take up exogenous dopamine. These findings are in concordance with studies on rabbit retinal preparations.