bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased adhesion response of anaplastic glioma cells to nerve growth factor and the presence of specific receptors
Authors: Vinores S. A.
Published: January 2005

A trypsin‐degradable nerve growth factor (NGF) receptor associated with the phospholipid component of the surface membrane has been detected on F98 anaplastic glioma cells. NGF also bound to the nucleus of F98 cells. Bound NGF was not displaceable by insulin, cytochrome C, growth hormone, or bovine serum albumin. Specific binding of NGF occurred with a Kd of 8.79 x 10−12 M as determined by Scatchard analysis with approximately 34,000 receptors per cell. Specific NGF binding was also evident to C6 rat glioma cells and IMR‐32 human neuroblastoma cells, but not to 3T3 mouse fibroblasts. These observations coupled with previous findings suggest that the NGF receptor may be a marker found on cells of neural derivation.

As little as 1 ng/ml NGF caused an increase in the adhesiveness of F98 cells to culture flasks. Increased adhesiveness could be observed in as little as 5 min and was apparent for at least 45 min. At 25 min in NGF‐containing medium, 24 ± 3% of the cells adhered to the flasks compared to 13 ± 1% of control cells. The NGF‐induced increase in adhesiveness was not duplicated by epidermal growth factor, insulin, cytochrome c, bovine serum albumin, dibutyryl cyclic AMP, or sodium butyrate. Oxidized NGF blocked the effect of native NGF, but had little or no adhesion‐promoting activity itself. Pretreatment of the cells with NGF was also effective in promoting adhesion, even though nerve growth factor was not added to the binding medium. The effect of this pretreatment was reversible; when NGF‐pretreated cells were grown in medium without supplemental NGF, the adhesiveness of the cells returned to control levels or lower.
Modulation of protein synthesis in a cell‐free system derived from rat brain by corticotropin (ACTH), magnesium, and spermine
Authors: Schrama L. H. , Edwards P. M. , Schotman P.
Published: January 2005

Modulation of protein synthesis by fragments of the ACTH molecule has been studied in a cell‐free system obtained from subcortical brain tissue of rats. Both the activity of the protein‐synthesizing system and its sensitivity to ACTH‐like peptides appeared to be highly dependent on the Mg2+ and spermine concentrations. At optimal Mg2+ concentrations (4 mM) the peptide sequences ACTH(1–24) and (11–24) were both inhibitory, the latter being the more active. The inhibitory effect was reduced or abolished at higher (suboptimal) Mg2+ concentrations.

Spermine, like ACTH, inhibited protein synthesis at the optimal Mg2+ concentration. However, at lower Mg2+ concentrations spermine had a stimulatory effect and maximal activity was obtained at 0.75–1.0 mM Mg2+. In the presence of spermine (60 μM) and Mg2+ (0.75 mM), a half‐maximal inhibition of protein synthesis was obtained with a peptide concentration of 5 μM. A structure‐activity study showed that the peptides ACTH(7–16)‐NH2, (11–24), (5–18, 17Lys18Lys)‐NH2 and (15–24) were active in inhibiting protein synthesis, whereas the fragments ACTH(1‐16)‐NH2 and (17–24) were inactive. The results are discussed in terms of an interaction between ACTH, Mg2+, and spermine, and intracellular processes involved in protein synthesis.
Covalent attachment of 125I‐β nerve growth factor to its receptors on sympathetic neurons
Authors: Stach R. W. , Rusenko K. W.
Published: January 2005

When 125I‐β nerve growth factor binds to sympathetic and sensory neurons, some labeled ligand is sequestered (becomes inaccessible to the external milieu) in a time‐ and energy‐dependent manner. It would appear that the higher affinity receptor (type I) participates in this process to a greater extent than does the lower affinity receptor (type II) [Olender and Stach, 1980; Olender et al., 1981]. A small portion of the sequestered 125I‐β nerve growth factor is found as part of a high molecular weight complex. When cells, which have been incubated with 125I‐β nerve growth factor, are solubilized with Triton X‐100 and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, a complex with an apparent molecular weight of approximately 240,000 is obtained. The formation of the covalent complex can be prevented by the prior addition of excess unlabeled β nerve growth factor or sodium fluoride and dinitrophenol. The covalent 125I‐β nerve growth factor‐receptor complex is dissociated in 50 mM dithiothreitol indicating that disulfide linkages are involved. At concentrations of β nerve growth factor (3.8 × 10−11 −3.8 × 10−10 M) where maximal fiber outgrowth occurs in vitro, approximately 50‐266 attomoles (0.3‐1.6% of the type I receptors) of the covalent complex are formed per 107 nerve cells. These data suggest that a small portion of the 125I‐β nerve growth sequestered by sympathetic neurons becomes covalently attached to its receptor subsequent to its sequestration in a manner which appears to involve type I receptors.
Sodium transport in astrocytes
Authors: Walz Wolfang , Hertz L.
Published: January 2005

Sodium transport in astrocytes in homogenous primary cultures from mouse brain cortex were investigated with radiotracer (22Na) and electrophysiological methods. The equilibrated Na+ content was 190 nmol × mg−1 protein and the influx and efflux rates were identical at about 560 nmol × mg−1 × min−1. No significant change was observed in Na+ efflux or influx when external K+ was raised from 5.4 to 12 or 54 mM, but the Na+ content decreased. Intracellular Na+ loading, evoked by previous exposure to ice‐cold K+‐free medium, doubled the Na+ efflux. Ouabain, a Na+ ‐K+ exchange inhibitor, exerted a small, nonsignificant inhibition of Na+ efflux at both 5.4 and 12 mM K+ and caused a large increase in Na+ content. At 5.4 mM K+, amiloride, a Na+‐H+ exchange inhibitor, decreased both influx and efflux of Na+ and caused an increase in Na+ content. Furosemide, an inhibitor of a cation‐Cl− carrier, decreased both content and influx of Na+ slightly but had no significant effect on Na+ efflux. The effects of amiloride or furosemide on Na+ influx were abolished at elevated (12 and 54 mM) K+. Attempts to stimulate the Na+−K+ pump with elevated external K+ or internal Na+ produced no electrogenic component of the membrane potential. Probably owing to the high K+ permeability. Based on the present results and earlier experiments on K+ influx, it is concluded that 1) the Na+−K+ pump of astrocytes under normal conditions transports more K+ than Na+; 2) intracellular Na+ loading increases Na+ efflux; 3) some Na+−H+ exchange and cotransport of Na+ and Cl− seem to occur at 5.4 mM K+; and 4) neither of the latter two transport mechanisms is enhanced at elevated K+ concentrations.
A brain synaptic dopamine‐binding protein: Isolation and partial characterization
Authors: Moroi K. , Hsu L. L.
Published: January 2005

A dopamine‐binding protein (DABP) has been purified from the rat brain cortex to homogeneity. Solubilization of the DABP from the synaptosomal membranes (P2M) by cholic acid, subsequent agarose gel filtration of the cholic acid extract to separate phospholipids from the DABP, and lastly DA affinity chromatography successfully resulted in a purified DABP with approximately 0.006% yield in protein concentration and 0.03% yield in specific [3H]‐DA binding. The specific [3H]‐DA binding of the purified DABP was 117 fmol/mg protein/10 min with a 4.6‐fold purification compared with the whole homogenate. The purified DABP had an Rf value of 0.67 on native disk polyacrylamide gel and it gave one single polypeptide subunit on the SDS gel with an Rf value of 0.63. The apparent molecular weight of this single subunit was estimated to be 34.5 kilodaltons. The elution patterns from either DA‐ or ADTN‐affinity (2‐amino‐6, 7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene‐affinity) columns indicated that this DABP had higher affinity for DA agonists than for DA antagonists. Photoaffinity labeling of [3H]‐DA to this DABP in the P2M fraction and the specific [3H]‐DA to the purified DABP demonstrated a nanomolar range affinity corresponding to either D2 or D3 receptors. These data suggested that the purified DABP could be related to either D2 or D3 receptors in the brain.
Metabolism and function of gangliosides in developing neurons
Authors: Dreyfus H. , Ferret B. , Harth S. , Gorio A. , Durand M. , Freysz L. , Massarelli R.
Published: January 2005

Previous experiments have shown that the addition of a mixture of gangliosides to the growth medium induced morphological changes in primary neuronal cultures, producing especially a trophic effect and a sprouting of neurites (neuritogenesis). The study reported here examined the changes of some biochemical parameters that paralleled the morphological modifications of cultured neurons from chick brain hemispheres treated with gangliosides. Neurons cultured from 3 to 7 days in the presence of various concentrations of a purified mixture or of single‐species of gangliosides (GM1, GD1a, GT1b) revealed that these glycolipids were easily incorporated into the cells as a function of their exogenous concentrations. Incubation of neurons with N‐acetyl‐D‐[U‐14C]mannosamine showed a final labeling of all endogenous cellular and exogenous incorporated gangliosides; however, the radioactivity recovered decreased as a function of the number of sialic acid units of the exogenously added gangliosides. The treatment of neuronal cells from 3 to 7 days in culture with a mixture of 10−8 M and 10−5 M gangliosides led to the following observations on some neurochemical parameters: no effect on the influx of choline and dopamine; no effect on the spontaneous choline efflux, whereas the K+‐provoked one is abolished; decrease of the spontaneous and K+‐stimulated release of dopamine; no effect on the spontaneous release of GABA for 10−8 M gangliosides but an increase of both spontaneous and K+‐provoked release for 10−5 M gangliosides. The data suggest that the possible insertion of gangliosides into the neuronal membranes may imply structural modifications that may influence enzymatic activities, neurotransmitter transport, and finally, some nerve cell mechanisms.
Can functional reorganization of area 17 following monocular deprivation be modified by GM1 internal ester treatment?
Authors: Carmignoto G. , Canella R. , Bisti S.
Published: January 2005

It has been extensively reported that monocular exposure early in life leads to profound alterations in visual cortical areas, where the majority of cells become responsive only to the stimulation of the normal eye. We have investigated a possible effect of the monosialoganglioside internal ester, termed AGF2, on the neuronal cortical plasticity of the kitten's visual cortex following monocular deprivation. Results indicate that in monocularly deprived kittens treated with ganglioside the ocular dominance shift in favor of the normal eye is partially prevented.
Inhibition of brain tubulinyl‐tyrosine carboxypeptidase by endogenous proteins
Authors: Modesti N. M. , Argaraña C. E. , Barra H. S. , Caputto R.
Published: January 2005

When a 25–50% ammonium‐sulphate‐insoluble fraction from a bovine brain preparation was chromatographed on a cellulose phosphate column, several protein fractions which inhibit the activity of tubulinyl‐tyrosine carboxypeptidase were obtained. One of these fractions exhibited activity of fructose‐bisphosphate aldolase (EC 4. 1. 2. 13) and the enzyme accounted for more than 95% of the protein of this fraction as judged by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. The inhibitory activities of the two protein fractions which had the highest activity per mg of protein were practically abolished by pretreatment with pronase; preincubation with trypsin, on the other hand, caused only a partial inactivation of the inhibitors. The inhibitory activities were little affected by heating at 90°C for 5 min. Preincubation with purified tubulinyl‐tyrosine carboxypeptidase caused a great decrease of the inhibitory activities of these two fractions, leaving open the possibility that these inhibitors act as substrates of the carboxypeptidase.
Growth cones and the formation of central and peripheral neurites by sensory neurones in amphibian embryos
Authors: Roberts A. , Patton D. T.
Published: January 2005

We have examined the primary growth of neurites by Rohon‐Beard sensory neurones in embryos of Xenopus, Rana, and Ambystoma. Using transmission electron microscopy we have confirmed that some central longitudinal neurites first grow directly under the basal lamina which surrounds the spinal cord in Xenopus embryos. Using scanning electron microscopy of dissected embryos we have followed the growth of neurites from the spinal cord to their target tissue, the skin. Comparisons between the three genera are made and detailed observations on growth cones during fasciculation, on the somites and on the basal lamina of the skin are presented. The observations are discussed in relation to possible influences on the course of growth of the developing neurites.
Polyribosomes under developing spine synapses: Growth specializations of dendrites at sites of synaptogenesis
Authors: Steward O. , Falk P.M.
Published: January 2005

We have previously reported that there is a dramatic increase in polyribosomes associated with dendritic spines during periods of synapse growth induced by denervating lesions. We suggested that polyribosomes at the postsynaptic site may somehow be involved in the growth of synapses. To evaluate this hypothesis further, the present study determines whether synapses which are growing in the developmental period also have accumulations of polyribosomes. We examined the dentate gyrus of the developing rat electron microscopically at 7, 10, 15, 20, and 28 days of age, which spans the major period of synaptogenesis in this structure. Qualitative observations revealed dramatic accumulations of polyribosomes under spine synapses in the youngest animals (7 and 10 days of age). With synapse development, the accumulations of polyribosomes became less dramatic, so that by 28 days of age, the neuropil of the dentate gyrus appeared qualitatively mature. To determine the relationship between polyribosomes under spine synapses and synapse development, quantitative electron microscopic methods were use to evaluate synapse density (number of synapses/100 μm2), and the incidence of polyribosome‐containing spines (proportion of spine synapses with underlying polyribosomes) in the neuropil of the dorsal blade of the dentate gyrus at each age. An inverse relationship was found between synapse density and the proportion of spines with polyribosomes. Synapse density increased in an almost linear fashion between 7 and 28 days of age to levels which were actually somewhat higher than in mature rats, whereas the incidence of polyribosome‐containing spines was highest at the youngest ages and decreased with development. Thus, polyribosomes were most prominent under spine synapses during the period of maximal synapse growth. These results, together with our previous observations of increased numbers of polyribosomes under spines during lesion‐induced growth, suggest that the polyribosomes represent a structural specialization of dendrites at sites of synapse construction. We propose that they produce protein(s) that are involved in synapse growth.
Aromatic L‐amino acid decarboxylase, dopamine β‐hydroxylase, monoamine oxidase, malondialdehyde, and acid phosphatase in rat brain capillaries and kidney glomeruli in experimental hypertension
Authors: Valli V.V. , Sadasivudu B.
Published: January 2005

Some metabolic consequences of experimental hypertension on rat brain capillaries and kidney glomeruli have been studied in rats made hypertensive by (a) a combination of deoxycorticosterone acetate injection and elevated salt intake (DOCA‐salt hypertension) and (b) isoproterenol injection. Enzyme activities were studied in vitro to ascertain directly or indirectly any changes in the metabolism of catecholamines and prostaglandins, and lysosomal integrity under conditions of experimental hypertension. Experimental hypertension was accompanied by an elevation in the activities of aromatic L‐aminoacid decarboxylase, dopamine β‐hydroxylase, and malondialdehyde concentration, both in the brain capillaries and kidney glomeruli of rats. On the other hand, monoamine oxidase activity increased in brain capillaries but decreased in kidney glomeruli. Acid phosphatase activity increased marginally in kidney glomeruli but decreased significantly in brain capillaries.

The catecholamine‐synthesizing potential appears to have been augmented in both the tissue capillaries with a compensatory increase in the degrading enzyme activity in the brain capillaries of hypertensive rats. The absence of such an increase and an actual decrease in the monoamine oxidase activity in the kidney glomeruli may be responsible for the sustained maintenance of the hypertensive state. Increased malondialdehyde concentration may be due to the stimulation of the prostaglandin metabolism by the augmented catecholamine metabolism.
Metabolically derived aspartate–elevated extracellular levels in vivo in iodoacetate poisoning
Authors: Sandberg M. , Nyström B. , Hamberger A.
Published: January 2005

Glycolysis was blocked in the hippocampus of the otherwise unaffected rabbit by perfusion of a previously implanted thin dialysis tube with a medium containing 0.1 mM iodoacetate. The perfusate from the dialysis tube was analyzed for free amino acids with liquid chromatographic separation of ortophthaldialdehyde derivatives. The data were used to calculate approximate levels of extracellular amino acids. Within 15 min after the beginning of iodoacetate perfusion, extracellular aspartate increased measurably, and the increase was tenfold within less than 2 hr. Although most other amino acids were unaffected, an elevation of taurine, phosphoethanolamine, and ethanolamine was observed with a delay of 30–40 min after the effect on aspartate.
Postnatal taurine deficiency in the kitten results in a persistence of the cerebellar external granule cell layer: Correction by taurine feeding
Authors: Sturman J.A. , Moretz R.C. , French J.H. , Wisniewski H.M.
Published: January 2005

Dietary deprivation of taurine in pregnant cats from approximately 1 week prior to giving birth is sufficient to reduce substantially the taurine concentration in feline milk but does not result in any abnormalities in kittens at birth. Kittens nursing on this low taurine milk have a lower growth rate than normal, have lower tissue taurine concentrations, and 8 weeks after birth have a persistence of cells in the cerebellar external granule cell layer. Mitotic figures are present also, indicating that cell division is occurring still, an event which normally is completed 3–4 weeks after birth. Daily oral supplementation with 40 μmoles taurine increases the growth rate almost to the level of normally nurtured kittens and results in normal tissue taurine concentrations and apparently normal migration of cells in the cerebellum. These findings indicate that nutritional taurine supplied in the milk is involved in the normal ontogeny of the cerebellum and that a taurine deficiency at this stage of development results in a maturational delay.
Ganglioside patterns during cerebral development in the normal and reeler mouse
Authors: Irwin L.N. , Hunter G.D. , Crandall J.E. , McCluer R.H.
Published: January 2005

Gangliosides were extracted from cerebral tissue of reeler and normal mice, and analyzed by two‐dimensional thin‐layer chromatography at embryonic and postnatal ages. The ganglioside pattern changed substantially as development proceeded in the telencephalon of both reeler and normal mice, but was the same at any given age for both conditions, despite the marked histological differences between reeler and normal samples. These results indicate that abnormal ganglioside patterns do not result from the reeler mutation at early stages of brain development, and that the cell misalignment characteristic of the reeler phenotype involves molecules other than gangliosides.
Thoracic dorsal funicular lesions affect the bouton patterns on, and diameters of, layer VB pyramidal cell somata in rat hindlimb cortex
Authors: Ganchrow D. , Bernstein J. J.
Published: January 2005

The effect of spinal dorsal funicular lesions (T 12) upon the frequency of boutons on, and diameters of the somata of pyramidal cells in layer VB of hindlimb cortex was studied. Adult rats sustained bilateral damage to either the dorsal column (DC, n = 10) alone or DC combined with the corticospinal tract (CS) (DC+CS, n = 34) and were utilized 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperatively (DPO). Neurons randomly sampled from 44 lesioned and 13 unoperated cases were analyzed for the number of silver‐impregnated boutons (Rasmussen method) on the circumference of the soma as well as diameters of the soma, nucleus, and nucleolus. Analyses of variance comparing across lesioned and normal groups were significant for bouton counts on the soma (P < 0.01), and diameters (long axis) of somata (P < 0.01) and their nuclei (P < 0.05). Both lesioned groups exhibited significant decreases from normal for these latter three parameters. With respect to survival time for the DC+CS‐lesioned animals we noted the following: (1) Bouton counts on the soma significantly decreased below normal between 1 and 60 DPO; this decrease was most dramatic during the first three days postlesion. (2) Somal diameter (long axis) significantly decreased below normal between 2 and 120 DPO (except at 14 and 90 DPO). (3) Nuclear diameter (long axis) significantly decreased below normal only at 90 DPO. (4) Bouton counts on somata of neurons in layers VB and IV [Ganchrow and Bernstein, 1981] of hindlimb cortex correlated negatively and significantly across 120 postlesion days. The rapid shrinkage and reduced afferentation of layer VB somata during the first week following DC+CS lesions suggest initial, retrograde reactions to CS axotomy. Since bouton counts on layer VB somata were significantly less (P < 0.05) in DC‐ than DC+CS‐lesioned rats, it is hypothesized that CS axotomy regulated a set‐point for increased afferentation which was maintained on the shrunken somata between 7 and 120 DPO.
Reactions of the geniculate cells to extraocular proprioceptive activation in rabbits
Authors: Molotchnikoff S. , Casanova C.
Published: January 2005

The aim of these investigations was to advance our knowledge of the influence of extrinsic ocular muscle (EOM) stretching on cell excitability of the retinocortical pathway. The initial goal was the lateral geniculate; however, histologic analysis indicated that most cells that responded to stretching were located near its superior edge, in an area analogous to the perigeniculate. Rabits were anaesthetized and prepared for single‐cell recordings. The extraocular muscles were detached from the eye and attached to a rigid tungsten hook. The hook was soldered to the pivot of a galvanometer that was controlled by a waveform generator. Precise and repeated length changes were obtained with extension and relaxation ramps of constant velocity. Thirty percent of the unit (N = 250) responded to EOM stretching. These stimuli evoked either an excitation or a decrease of the units' spontaneous activity. Several experimental controls provided evidence that the signals that evoked the geniculate responses originated from proprioceptors belonging to the EOM. Most responding cells had their receptive fields located eccentrically (≥50°). Also, cells that reacted to EOM stretching responded to optic nerve stimulation with a significantly shorter latency than cells that were unresponsive. Pairing the EOM stretching with light stimuli produced the following results. In about half of the units the light‐evoked responses augmentded, but in 28% of the cells the light‐evoked discharges decreased. These effects were obtained even though EOM stretching delivered singly failed to elicit a reaction from the cell. Histologic reconstructions indicated that cells were distribution in a discrete region lying rostral and dorsomedial to the lateral geniculate nucleus. This area has been associated with the perigeniculate nucleus in cats and rats.
Substrate oxidation by isolated rat brain mitochondria and synaptosomes
Authors: Tildon J. T. , Roeder L. M. , Stevenson J. H.
Published: January 2005

The rates of [6‐14C]‐glucose oxidation by reconstituted systems of cytosol and mitochondria or cytosol and synaptosomes were essentially the same as the rate of oxidation of [3‐14C]‐3‐hydroxybutyrate. However, the rate of [U‐14C]‐glutamine oxidation by mitochondria was 2.5 times that by synaptosomes. The addition of glutamine (5 mM) caused a reduction in the rates of oxidation of [6‐14C]‐glucose of 20% and 40% by mitochondria and synaptosomes, respectively. Conversely, the addition of glucose (5 mM) had little or no effect on the rate of [U‐14C]‐glutamine oxidation by either organelle. Amino‐oxyacetate decreased [U‐14C]‐glutamine oxidation by mitochondria more than 35% but had little or no effect on the rate of glutamine oxidation by synaptosomes. When glucose (5 mM) was added to [3‐14C]‐3‐hydroxybutyrate, the rates of oxidation by the mitochondria and synaptosomes were increased by 65% and 77%, respectively. However, in the reverse situation the addition of 3‐hydroxybutyrate decreased [6‐14C]‐glucose oxidation by synaptosomes (35%) but did not decrease the rate by mitochondria. These results suggest that differences in the rates of substrate utilization by mitochondria and synaptosomes and differences in substrate interactions in these two subcellular organelles may contribute to metabolic compartmentation in the brain.
Selected nutrients reduce the pyruvate requirement for survival in vitro of chick central nervous system neurons
Authors: Facci L. , Skaper S. D. , Varon S.
Published: January 2005

Central nervous system neurons, cultured as monolayers at low density, need exogenous pyruvate for their survival. The pyruvate concentrations required by embryonic day 8 (E8) chick forebrain neurons are reduced substantially by other low molecular weight agents present in Dulbecco's modified Eagle's medium (DMEM) and in astroglia‐conditioned Eagle's basal medium (EBM). To examine the nature of these pyruvate‐sparing molecules, E8 chick forebrain neurons were cultured in EBM containing a concentration of pyruvate too low to support neuronal survival, unless sparing agents were supplied. This stragegy permitted the identification of the pyruvate‐sparing DMEM constituents, omission of which had led to neuronal loss. Pyruvate sparing required a mixture of (i) serine, (ii) pyrioxal (or riboflavin and biotin), and (iii) six essential amino acids: arginine, cystine, leucine, isoleucine, tyrosine, and tryptophan (each replaceable by corresponding amino acids, except tryptophan). All pyruvate‐sparing contributors were optimally needed at or near their DMEM concentrations.
Coexistence of high‐affinity uptake mechanisms for putative neurotransmitter molecules in chick embryo retinal neurons in purified culture
Authors: Pessin M. , Adler R.
Published: January 2005

We report here that high‐affinity uptake mechanisms for two or three putative neurotransmitter molecules coexist in many of the neurons present in glia‐free, purified neuronal monlayers from chick embryo retina. Replicate cultures were incubated with the tritiated forms of the amino acids gamma‐aminobutyric acid (GABA), taurine (TAU) and aspartate (ASP) either individually or in binary or in binary or tertiary combinations. Incubation conditions were those typically used to analyze high‐affinity uptake mechanisms. At the end of the incubation period the cultures were either lysed in water for measurement of intracellular radioactivity by liquid scintillation counting, or fixed in glutaraldehyde and prepared for autoradiography. Our results indicate that there is coexistence of uptake mechanisms for putative neurotransmitters in retinal cells. Biochemical measurements showed that, at the concentrations used in these experiments, two or more radioactive amino acids could be taken up simultaneously by the cultured populations without extensive inhibition. Moreover, the percentage of cells that appeared autoradiographically labeled in cultures exposed to two or more radioactive amino acids was less than the sum of the percentages of labeled cells when each amino acid was applied individually. Numerical analysis of the autoradiographs was carried out to determine the percentage of cells that coul take up only one, only two, or the three putative amino acid neurotransmitters under investigation. This analysis showed that approximately 20% of the neurons have only the mechanism for TAU, whereas very few if any neurons have high‐affinity uptake mechanisms for GABA alone or ASP alone. Our experiments have identified populations of cells that take up GABA and TAU (but not ASP) or ASP and TAU (but not GABA). Interestingly, we have not seen any neurons that can take up ASP and GABA and cannot take up TAU, although as many as 50% of the neurons can take up ASP, GABA, and TAU simultaneously. By showing simultaneous uptake of different putative neurotransmitters within individual neurons, our results emphasize the concept that the presence in a given neuron of a high‐affinity mechanism for a particular molecule is not a sufficient criterion to assign a neurotransmitter function to that molecule in that neuron.
Transplantaion of embryonic chick spinal cord into transection adult chicken into transection adult chicken spinal cords: A useful model for transplantation research
Authors: Grady M. S. , Steward O. , Jane J. A.
Published: January 2005

Adult chicken spinal cords were completely transected under direct visualization. One week later, embryonic chick spinal cords 6 days, 9 days, or 11 days old were implanted into the site of transection. The animals were allowed to survive for 2 months and then killed and the spinal cords were prepared for histologic analysis. The survival and outgrowth of the embryonic transplants were compared with regard to the age of the implant, and its effects on the host tissue. Six‐day embryonic tissue survival and integrated far better than 9‐day or 11‐day, and adult spinal cord subjacent to the early age embryo showed fewer degenerative changes. The chick embryo may provide a model for use in CNS transplantation.
Effects of nitrendipine on the voltage‐sensitive calcium channel in mammalian sensory neurons
Authors: Litzinger M. J. , Nelson P. G. , Pun R. Y. K.
Published: January 2005

We examined the ability of nitrendipine, a calcium antagonist believed to block the voltage‐sensitiv calcium channels in a number of excitable membranes, to affect the calcium spike duration and rate of rise (Dv/Dt) in 4‐week‐old cultured mouse dorsal root ganglion neurous. Concentrations of 10 nM‐1:0 μM intrendipine reduced but did not eliminate calcium spike as measured by the rate of rise. These results were obtained at either 22° rate of rise (Dv/Dt) in 4‐week‐old cultured mouse dorsal root ganglion neurous. Concentrations of 10 nM‐1:0 μM intrendipine reduced but did not eliminate calcium spike as measured by the rate of rise. These results were obtained at either 22° or 37°C in solutions containing 5 mM calcium. A similar lack of total block was seen with prolonged incubation in sodium‐free recording media containing 1 μM tetrodotoxin (TTX), 1.8 mM calcium, and 1 μM nitrendipine. Calcium spike duration was affected in an inconsistent manner by nitrendipine and other 1,4 dihydropyridines. Cobalt (10 mM) totally blocked the voltage‐dependent calcium mechanism as measured by either the spike duration or the rate of rise. We conclude that calcium influx through the voltage‐sensitive calcium channel in mouse dorsal root ganglion neurons is not blocked by even high concentrations of nitrendipine.
Rat brain α2‐pre‐ and postsynaptic receptors are different or differently modulated?
Authors: Cerrito F. , Preziosi P.
Published: January 2005

In order to get better characterization of α2‐pre‐ and postsynaptic noradrenergic receptors in the rat brain, we investigated the α2‐receptor changes which take place during a 12‐day treatment with the α2‐antagonists yohimbine (4 mg/kg) and mianserin (10 mg/kg). These treatments caused a significant increase in the sensitivity of hypothalamic synaptosomes to the inhibitory action of the noradrenergic agonist clonidine on the 3H‐noradrenaline release elicited by K depolarization. Frontal cortex α2‐autoreceptors were not affected by drug treatments. However, the 3H‐p‐aminoclonidine (3H‐PAC) bindig to membranes from hypothalamus or frontal cortex from treated animals was the same as in controls.

Changes in neural firing, elicited by the α2‐antagonists on noradrenergic neurons, could explain our results.

The presynaptic autoreceptors may thus become hypersensitive to counteract the enhanced neurotransmitter release in the hypothalamus, where the noradrenaline is accumulated at the synaptic cleft. In the frontal cortex, where it seems that only 5% of the noradrenergic terminals make synaptic contacts with postsynaptic elements, the α2‐autoreceptors are less sensitive to an enhanced neurotransmitter release. Alternatively, they have scarce functional importance because the noradrenaline release is effectively modulated by the inhibitory recurrent locus coeruleus collaterals.

At the postsynaptic level, the receptor down‐regulation might be prevented by chronic presence of the antagonist drug.

Thus the different behavior between pre‐ and postsynaptic α2‐receptors and between α2‐receptors of different brain areas may be ascribed to a different modulation rather than to different molecular arrangements.
Purification and characterization of cultures of oligodendroglia from rat brain
Authors: Poduslo S. E. , Curbeam R. , Miller K. , Reier P.
Published: January 2005

It is possible to purify dary cells from rat brain which by ultrastructural criteria are oligodendroglia. The modified technique involves the use of an isolation medium less damaging to the initial cell suspension and then differential plating of a stratified primary cell culture obtained from 1–3‐day‐old rat brain which has been described. Five percent serum encourages the growth of oligodendroglia. In this system, the oligodendroglia can be harested several times from the primary cultures. To retain the homogeneous populations of oligodendroglia, it is necessary to constantly manipulate the cultures by hitting the flasks and replating the suspended cells. Electron microscopy reveals that the small dark cells are differentiated oligodendroglia with prominent microtubules. Immunofluorescence staining of the cells shows that the majority of the cells are negative for known surface markers which include cerebrosides and 04 antigen (markers for oligodendroglia), glial fibrillary acidic protein, and fibronectin. Interestingly, it is possible to increase the number of 04‐positive cells by the addition of dibutyryl cyclic AMP to the medium. Rat oligodendroglia are able to incorporate about 20% of the radioactivity into cerebrosides, using the substrate, [3H]galactose; this is a biochemical feature unique to oligodendroglia. The expression of all the oligodendroglial antigens should be remarkably enhanced when the cells are induced to produce myelin.
Lipid and glycolipid metabolism of cultured astrocytes: A time course study
Authors: Yim S. H. , Monsma S. , Hertz L. , Szuchet S.
Published: January 2005

Primary cultures of astrocytes free of neurons and containing less than 1% of oligodendrocytes were examined for their ability to incorporate labeled precursors into lipids and glycolipids. At selected developmental stages cultures were doublelabeled with either [3H]glycerol and [14C]acetate or with [3H]galactose and Na2[35SO4] for a total of 72 hr. Lipids were extracted with CHCl3/CH3OH, fractionated on a silicic acid column, and further resolved by two‐dimensional thin‐layer chromatography. It was found that (1) cultured astrocytes actively incorporate acetate and glycerol into various phospholipids; (2) they have very limited ability to utilize galactose and virtually lack the synthetic machinery to use Na2SO4 for the synthesis of sulfated sphingogalactolipids; and (3) their overall lipid metabolism is very distinct from that of oligodendrocytes. It was also found that cultured astrocytes have low levels of 2′,3′ cyclic phosphodiesterase and glycerol phosphate dehydrogenase activities; the latter is less than one fifth of that in oligodendrocytes.
Glial differentiation in dissociated cell cultures of neonatal rat brain: Noncoordinate and density‐dependent regulation of oligodendroglial enzymes
Authors: Wernicke J. F. , Volpe J. J.
Published: January 2005

The mixed glial system of primary cultures of cells dissociated from neonatal rat brain was utilized to study glial differentiation. The investigation was addressed specifically to the possibility of noncoordinate regulation of two manifestations of oligodendroglial differentiation, i.e., activities of glycerol‐3‐phosphate dehydrogenase (GPDH) and of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), as well as the effects of initial cell density on the time of onset and the intensity of expression of these aspects of oligodendroglial differentiation. Simultaneously, glutamine synthetase activity was studied to determine effects on astrocytic differentiation. GPDH exhibited a major developmental increase in specific activity between 20 and 32 days in culture. However, CNP activity exhibited a major developmental increase that commenced approximately 2 weeks earlier. The onset of these expressions of oligodendroglial differentiation was not affected by such environmental factors as initial cell density. However, the intensity of expression of the temporally separate increases in GPDH and CNP activities was markedly density‐dependent. The highest activities were attained in cultures plated at the lowest cell densities. The astrocytic enzyme, glutamine synthetase, also exhibited a striking developmental increase (approximately tenfold between 13 and 30 days in culture), but initial cell density affected neither the time of onset nor the intensity of expression of this aspect of astrocytic differentiation. The data demonstrate a striking developmental increase in GPDH activity that is not coordinate with that in CNP. The noncoordinate manifestations of oligodendroglial differentiation commence as a function of time in culture, whereas the intensity of expression of this differentiation can be influenced by such environmental factors as initial cell density. Neither the onset of nor the extent of the sharp increase in glutamine synthetase activity, an expression of astrocytic differentiation, is affected by initial cell density.
The role of charge microheterogeneity of basic protein in the formation and maintenance of the multilayered structure of myelin: A possible role in multiple sclerosis
Authors: Moscarello M. A. , Brady G. W. , Fein D. B. , Wood D. D. , Cruz T. F.
Published: January 2005

Isolation of several of the charge isomers from both normal and multiple sclerosis (MS) myelin basic protein (MBP) was achieved on CM‐52 columns at pH 10.6. In liquid x‐ray diffraction and agregation experiments, corresponding charge isomers were equally effective in the formation of multilayers, demonstrating the dominant role of overall net positive charge. These studies demonstrated that the change in overall charge of MBP of one net positive charge was sufficient produce large changes in aggregation and in multilayer formation. The x‐ray diffraction experiments showed that component 1 was twice as effective as component 2 although they differed in charge by a single positive charge. Component 3 was less effective than component 2 and component “8” was not effective at all. Vesicle aggregation also showed a dependence on net positive charge. In order of decreasing effectiveness, component 1 > component 2 > component 3 > component “8”.

Since overall charge on MBP is determined by contributions from the various charge isomers, the relative proportions of these charge isomers favoring the less cationic components could explain the observation that MBP from MS victims was less effective than MBP form normal brain in vesicle aggregation and multilayer formation. The isolation of myelin‐containing white matter fractions from both normal and MS tissue in which the loss of some of the most cationic charge isomers was correlated with presence of less compact myelin supports this hypothesis.
Acidic lipids enhance cathepsin D cleavage of the myelin basic protein
Authors: Williams K. R. , Williams N. D. , Konigsberg W. , Yu R. K.
Published: January 2005

Some acidic lipids including sulfatide and phosphatidylinositol were found to increase greatly the rate of cathepsin D cleavage of the myelin basic protein. Since a similar effect was seen when the substrate was changed to cytochrome C, but not when the enzyme was changed to pepsin, these acidic lipids seem to be acting on cathepsin D rather than on myelin basic protein itself. Even so, chemical modification studies suggest that this phenomenon is only seen when the myelin basic protein is in its native conformation. Succinylation of MBP increases its rate of cleavage by cathepsin D by at least tenfold and, in addition, with this modified and presumably denatured MBP as substrate, activation of cathepsin D is no longer seen with acidic lipids. These findings suggest that the native conformation of MBP is both an important determinant of its rate of cleavage by cathepsin D and is also essential for observing activation of this reaction by acidic lipids. The acidic lipids seem to alter the “extended active site” of cathepsin D in such a way as to enable this enzyme better to utilize the native myelin basic protein as a substrate. Cathepsin D has previously been implicated as the protease responsible for the release into cerebrospinal fluid in multiple sclerosis patients of an encephalitogenic fragment derived from myelin basic protein. It is possible that the elevated levels of cathepsin D and sulfatide that have previously been found associated with multiple sclerosis plaques in vivo act in concert to bring about the rapid cleavage and subsequent loss of the myelin basic protein from these localized regions in the myelin sheath.
Reversal by estrogen of the effect of dorsal raphe stimulation on release of LH but not prolactin
Authors: Kitts C. S. , Johnson J. H.
Published: January 2005

Electrical stimulation of the dorsal raphe nucleus (DRN) was administered to rats 2–4 weeks after ovariectomy and at last 7 days following implantation of a bipolar electrode. During stimulation the concentration of luteinizing hormone (LH) decreased whereas that of prolactin (Prl) increased in serial plasma samples obtained from the unanesthetized animals via a cannula chronically implanted in the right external jugular vein. Administration of estradiol benzoate (5 μ/100 g BW) for 2 days prior to the experiment reversed the effect of stimulation of LH but augmented the Prl response, suggesting modulation by estrogen of the relative effectiveness of opposing pathways ascending from DRN to influence LH release. It is also apparent that LH and Prl release are controlled by distinct mechanisms since estrogen exerted opposite influences on the responses of these two hormones to stimulation of DRN.
High molecular weight microtubule‐associated proteins: Purification by electro‐elution and amino acid compositions
Authors: Kosik K. S. , Bakalis S. F. , Selkoe D. J. , Pierce M. W. , Duffy L. K.
Published: January 2005

The high molecular weight microtubule‐associated proteins (MAPs) consist of MAPs 1A, 1B, 1C, 2A, and 2B. The proteins have been prepared from rat brain by using taxol and resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The individual proteins were purified by electro‐elution from stained fixed gels and shown to migrate at their original mobility without proteolysis. Amino acid compositions of purified MAP 1 and 2 component proteins are reported and compared.
Purification and characterization of plasma membranes obtained from rat neurons prepared by bulk‐isolation
Authors: Poduslo S. E. , Chechik T. , Filbin M. T. , Rosenfeld J.
Published: January 2005

The properties of neuronla plasma membranes are highly specialized, since they include the receptors by which many hormones, neurotransmitters, drugs, and toxins initiate their actions, as well as the components necessary for receiving synaptic input from a variety of sources and for generating the appropriate pattern of action potentials. To be able to study plasma membranes of neurons, it is necessary to separate sufficient quantities of neurons from the other cell types, processes, and myelin found in brain. The methodology for obtaining neurons by bulk‐isolation from 10‐day‐old rat brain has been established, and techniques for purifying the plasma membranes are presented here. The procedure involves swelling the cells in a buffered solution, homogenizing the cells vigorously, and separating the membrane fragments on discontinuous sucrose gradients. The plasma membrane fraction is enriched in plasma membrane marker enzymes and has low activity for contaminating enzymes for subcellular organelles. Electron micrographs show that the fraction consists of membrane vesicles and profiles of various sizes. The protein composition reveals over 60 proteins with molecular weights as high as 100,000 to a low of 14,000. This plasma membrane fraction now can be the focus for studying receptors, glycoproteins, and cell‐specific markers of neurons in health and disease.
Effect of nicotine aerosol on slowly adapting receptors in the airways of the dog
Authors: Taylor R. F. , Lee L.‐Y. , Jewell L. A. , Frazier D. T.
Published: January 2005

The response of slowly adapting airway stretch receptors to nicotine aerosol was studied in the paralyzed, artificially ventilated, anesthetized dog. Single‐unit stretch receptor recordings were made from individual vagus nerve filaments placed on a pair of platinum hook electrodes. Administration of 2% nicotine aerosol for five consecutive breaths caused an increase in both the peak transpulmonary pressure (Ptp) and in the activity of the slowly adapting stretch receptors (SARs). The results suggest that tracheal SARs were more affected than those receptors located distal to the carina. Adminsitration of nicotine aerosols following pretreatment with isoproterenol, a bronchodilator, failed to significantly increase Ptp and, concomitantly, the activity of SARs. Therefore, the stimulatory effect of nicotine on SARs appeared to involve primarily an indirect activation of SARs vianicotine‐induced bronchoconstriction. It is suggested that the activation of SARs may be involved in the reported nicotine‐dependent cigarette smoke‐induced apnea.
Sequence homology between tyrosine hydroxylase mRNA and a rat adrenal medullary cDNA
Authors: O'Malley K.L.
Published: January 2005

A rat adrenal medullary recombinant clone has been isolated by cross‐hybridization with a tyrosine hydroxylase (TH) cDNA. The new clone has a mRNA size of 5.6 kb and also hybridizes to the 1.9‐kb tyrosine hydroxylase message. Southern blot analysis reveals several hybridizing bands in common between the TH cDNA and the adrenal medullary clone. These results demonstrate that the adrenal clone shares sequences in common with the TH gene and/or is closely linked to it in the genome. Hybrid‐selected mRNA translation products of the adrenal clone can be immunoprecipitated with dopamine beta hydroxylase antisera. This suggests that the adrenal medullary cDNA may code for another catecholamine pathway enzyme.
Gene expression in hypothalamic neurons: Luteinizing hormone releasing hormone
Authors: Pfaff D.W.
Published: January 2005

In situ hybridization allows the detection and measurement of specific messenger RNAs in individual hypothalamic neurons, and has shown, among magnocellular neurons, not only which cells express the genes for oxytocin and vasopressin but also how they change with physiological stimulation. With this technique, neurons expressing a gene for luteinizing hormone releasing hormone‐like messenger RNA have been discovered in the preoptic area and diagonal bands of the rat forebrain. Seven days of estrogen treatment of ovariectomized female rats increases the LHRH‐like messenger RNA in this neuronal system.