bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible role of increased brain methylation in methionine sulfoximine epileptogenesis: Effects of administration of adenosine and homocysteine thiolactone
Authors: Schatz R. A. , Wilens T. E. , Tatter S. B. , Gregor P. , Sellinger O. Z.
Published: January 2005

An intraventricular pulse of [14COOH]L‐methionine to mice pretreated with the convulsant L‐methionine‐dl‐sulfoximine (MSO) resulted in significantly higher than control specific radioactivity values of cerebral [14COOH]L‐methionine (Met), [14COOH]S‐adenosyl‐L‐methionine (AdoMet) and [14COOH]S‐adenosyl‐L‐homocysteine (AdoHcy). MSO administration (3 hr) also decreased brain steady‐state levels of Met, AdoMet, and AdoHcy.

Following an intraventricular pulse of [3H‐methyl]L‐methionine, the levels of [3H‐methyl]phosphatidylmonomethylethanolamine and of membrane associated and soluble [3H‐methyl]carboxylmethylated proteins were increased over corresponding saline‐treated controls. The activity of cerebral histamine N‐methyltransferase was also increased after MSO treatment. The administration of a combination of adenosine and homocysteine thiolactone to MSO‐pretreated animals counteracted the MSO‐induced decreases in brain Met, AdoMet, and AdoHcy as well as the increase in histamine N‐methyltransferase activity. In addition, administration of adenosine together with homocysteine thiolactone decreased the incidence of, and increased the latency to MSO seizures, with the most effective anticonvulsant action occurring when cerebral AdoHcy levels were at their highest.
Glial contribution to amino acid content and metabolism of the deafferented dentate gyrus
Authors: Molin S.‐O. , Nyström B. , Haglid K. , Hamberger A.
Published: January 2005

The time course of tissue content and evoked release of endogenous amino acids was analyzed in the partially deafferented dentate gyrus of the rat hippocampus 2– 24 days following unilateral lesion of the perforant path. Amino acids in tissue extracts and perfusates were determined after precolumn derivatization and hplc separation. The astrocytic glial cell reaction was monitored with immunohistochemistry of S‐100.

The tissue content of glutamate decreased significantly on the lesioned side, whereas only a moderate reduction in taurine, aspartate, and alanine occurred. Glutamine was significantly elevated at 7 days. The evoked efflux of glutamate was reduced at 2 and 7 days, whereas no change was seen at longer survival periods. The evoked release of GABA and aspartate increased on the denervated side after 12 and 24 days. The rate of carbon utilization into amino acid pools was followed with 14C‐glucose and 14C‐acetate. The incorporation of acetate showed a peak 2–9 days following lesion, which paralleled in time the hypertrophic glial cells. The incorporation of glucose decreased during this period. The metabolic events are discussed in relation to the morphological changes in synapses and glial cells.
Histochemical demonstration of cytoplasmic glycosaminoglycans in the macroneurons of the human central nervous system
Authors: Alvarado M. V. , Castejón H. V.
Published: January 2005

The presence of glycosaminoglycans (GAG) has been histochemically demonstrated in the CNS of various mammalian species. They have been related with some nerve functions as neurotransmitters storage and synaptic transmission. In the present paper, the histochemical properties of nerve cell cytoplasmic GAG were studied in several regions of adult human CNS. Samples of brain cortex, pons, upper medulla, and cerebellar cortex obtained by autopsy from subjects not dying after neurological diseases were fixed by immersion in glutaraldehyde, dehydrated with ethanol, and embedded in paraffin. The sections were stained with Alcian blue solutions adjusted to pH 2.5, 4.0, and 5.7. To the latter solution MgCl2 was added in increasing concentration from 0.05 to 1.2 M. Testicular hyaluronidase, neuraminidase, and ribonuclease were applied on simultaneous sections with their respective controls. The sequence of these reactions allowed us to demonstrate the presence of hyaluronic acid along chondroitin‐4‐ and/or 6‐sulphate in the cytoplasm of most nerve cells. The sulphated GAG showed certain variability in the various regions studied related specially with their grade of sulphation.
A combined flat‐embedding, HRP histochemical method for correlative light and electron microscopic study of single neurons
Authors: Aldes L. D. , Boone T. B.
Published: January 2005

A simple and reproducible method is described for studying the morphology of the same neuron at the light and electron microscopic level. This method utilizes horseradish peroxidase histochemistry and a recently described flat‐embedding procedure wherein thin, aldehyde‐fixed sections are placed in resin between glass microscope slides pretreated with dimethyldichlorosilane. The significance of these combined methodologies for correlative light and electron microscopic studies of single neurons is discussed.
Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland
Authors: Champney Thomas H. , Holtorf A. P. , Steger R. W. , Reiter R. J.
Published: January 2005

This study details the procedures involved in measuring a number of the components of indole metabolism within the pineal gland. Tryptophan, 5‐hydroxytryptophan, serotonin, N‐acetylserotonin, melatonin, 5‐hydroxyindoleacetic acid, N‐acetyltransferase (NAT) activity, and hydroxyindole‐O‐methyltransferase activity were measured by a combination of three techniques, which included microassays, radioimmunoassay, and high‐performance liquid chromatography. Determination of the melatonin synthetic pathway components within the same gland reduces the number of animals needed for studies and allows correlations between these constituents to be calculated. Very high degrees of correlation (r=0.91−0.99) are seen between those compounds which exhibit significant rhythms (serotonin, NAT activity, N‐acetylserotonin, and melatonin) when group means are compared. When correlations are calculated on a per‐animal basis throughout the experiment, moderate to high degrees of correlation (r=0.57−0.79) are found among those components that exhibit rhythms. However, when correlations are determined on a per‐animal basis at each time point, no significant correlations are found. One hypothesis accounting for these differences may be that changes occur in indole metabolism within the same pineal gland over a period of a few minutes.
Ionic mechanism of a hyperpolarizing glutamate effect on two identified neurons in the buccal ganglion of Aplysia
Authors: Sawada M. , Hara N. , Ito I. , Maeno T.
Published: January 2005

The ionic mechanism of a membrane effect of L‐glutamate on two identified neurons in the buccal ganglion of Aplysia kurodai was investigated with conventional microelectrode techniques and glutamate iontophoresis. Bath‐applied and iontophoresed glutamate hyperpolarized the membrane and increased the membrane conductance. The hyperpolarizing glutamate response decreased in amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of the hyperpolarizing glutamte response was close to the ECl (−60 mV). The reversal potential changed by 22.4 mV when the external chloride concentration was altered by a factor of 5. The relationship between the iontophoretically applied current and the membrane conductance changes was suggestive of two glutamate molecules reacting with a single receptor site. The hyperpolarizing glutamate response was essentially unaffected by 2‐amino‐4‐phosphonobutyric acid (2‐APB), L‐proline, and quinuclidinyl benzilate (QNB). It was concluded that the hyperpolarizing glutamate response was generated by an activation of Cl− conductance.
Glial cells dissociated from newborn and aged mouse brain
Authors: Vernadakis A. , Mangoura D. , Sakellaridis N. , Linderholm S.
Published: January 2005

Changes occurring with days in culture and cell passage in cultured glial cells derived from newborn vs aged (18‐mo) mouse cerebral hemispheres were compared. The activities of the enzymes glutamine synthetase (GS), an astrocyte marker, and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), an oligodendrocyte marker, were determined. In addition, glial fibrillary acidic protein (GFA) and glycerol phosphate dehydrogenase (GPDH) immunoreactivity was used to morphologically identify astrocytes and oligodendrocytes, respectively. In cultures derived from newborn mouse cerebral hemispheres, both GS and CNP activity and GFA‐positive and GPDH‐rhodamine‐positive cells were present with cell passage. In general, GS activity did not change in early cell passage in cultures from either newborn or aged mouse; in passage 5, GS was high in both sources of cell populations. CNP activity increased with cell passage in cultures derived from newborn mouse; in cultures derived from aged mouse CNP was low in the primary cultures, increased with cell passages 2 and 3, and declined with passages 4 and 5. The survival of astrocytes as shown by GS and the decline in oligodendrocytes as shown by CNP was also supported by an increase in the proportions of GFA and GPDH immunoreactive cells. We interpret the increase in GS activity to parallel the astrogliosis observed in vivo in the aging brain. Moreover, the decline in oligodendrocytes in culture may represent a shift of balance between glial cell types that appears to be influenced by the age of brain tissue and time in culture.
Vitamin content of some normal human brain segments
Authors: Baker Herman , Frank O. , Chen T. , Feingold S. , De Angelis B. , Baker E.
Published: January 2005

Nicotinates, pantothenates, riboflavin, vitamins B6 and B12′ free (acetyl) and total (free and bound) choline, biopterin, thiamin, biotin, methylated and nonmethylated folates in frontal, temporal, precentral, postcentral, and occipital cortex, thalamus, cerebellum, pons, basal ganglia, and substantia nigra were estimated. Nicotinates are significantly more concentrated in basal ganglia and thalamus than pons. Nonmethylated folate content is not significantly varied in brain segments; the pons contains more methylated folate. Riboflavin content is higher in the basal ganglia and temporal cortex than frontal cortex. Biotin is concentrated in pons and basal ganglia. Thiamin concentration is less in the postcentral cortex than the thalamus and substantia nigra. Biopterin is significantly higher in substantia nigra and basal ganglia than the other brain segments. Total choline content is high in substantia nigra, pons, and thalamus; free (acetyl) choline is significantly elevated in basal ganglia. B12 content is less concentrated in the cortex segments. B6 is highly concentrated in the basal ganglia. Pantothenate content is elevated in pons when compared to the various cortex segments and cerebellum.
Effects of exogenous GM1 and GD1a on S20Y neuroblastoma cells
Authors: Carine K. , Schengrund C.‐L.
Published: January 2005

The effects of exogenous GM1 and GD1a on S20Y murine neuroblastoma cells were assessed by monitoring morphology, tumorigenicity, mitotic index, and plating efficiency. S20Y cells were seeded at a density equivalent to 5 × 104 cells per 35‐mm tissue culture dish; 38–42 hr after seeding (preconfluent stage) the cells were treated for 12 hr with 100 μg of ganglioside per ml of medium in which the serum content was reduced from 10% to 0.5%. Analysis of the cell lipids indicated that added ganglioside became tightly associated with the membrane during the 12‐hr exposure. GM1 treatment resulted in increased projections on the cell surface and fine structures projecting from the cell processes. GD1a treatment resulted in a reduction in the cellular mitotic index. Plating efficiency was reduced by both GM1 and GD1a. Neither ganglioside affected tumorigenicity of the S20Y cells. Twelve hours after removal of the added ganglioside and exposure of the cells to normal medium, the ganglioside composition of the membranes from treated cells approached that of the controls, and the ganglioside‐induced effects had been reversed. These results suggest that addition of specific gangliosides induces different cellular responses and that these changes are dependent upon the continued presence of the ganglioside.
Inhibition of thalamic ventrobasal complex neurons by glutamate infusion into the thalamic reticular nucleus in rats
Authors: Mushiake S. , Shosaku A. , Kayama Y.
Published: January 2005

In urethane‐anesthetized rats a 0.36‐mm metallic cannula for infusion was positioned in the somatosensory component of the thalamic reticualar nucleus (sTR), where movement of the vibrissae evoked neuronal discharge. Infusion there of 0.125–0.5 μl of a 50 mM solution of glutamate over a 1‐min period suppressed both spontaneous and evoked discharge of neurons in the ventrobasal complex (VB), but only for those which also responded to vibrissal stimulation. VB neurons activated by somatosensory stimuli at other locations were unaffected. Thus, excitation of neurons in sTR inhibits those in VB, but the effect appears to be highly coordinated somatotopically.
Serotonin uptake and release from rat cerebellum in vitro
Authors: Beas‐Zarate C. , Sandoval M. E. , Feria‐Velasco A.
Published: January 2005

Serotonin (5‐HT) uptake and release were analyzed in cerebellar fractions. Cerebellar P2 fractions and molecular layer homogenates are able to accumulate exogenous 5‐HT through an Na‐dependent, high‐affinity transport system. Molecular layer 5‐HT uptake, however, was totally dependent upon Na+, whereas the accumulation of the amine by P2 fractions from whole cerebellum was also carried out by an Na‐independent transport system. Ca++‐dependent release of recently accumulated 5‐HT was observed in both P2 fractions and molecular layer homogenates when depolarizing conditions were used. This release was blocked by ruthenium red and Mg++, two inhibitors of the Ca++ entry to the nerve endings induced by depolarization. The molecular layer homogenates released twice as much 5‐HT as did cerebellar P2 fractions, indicating that serotoninergic boutons in the cerebellar cortex might be located in the molecular layer. Our results suggest that 5‐HT might play a transmitter role in the cerebellum.
Analytical and preparative high‐performance liquid chromatography of gangliosides
Authors: Gazzotti G. , Sonnino S. , Ghidoni R. , Kirschner G. , Tettamanti G.
Published: January 2005

Analytical and preparative procedures are described for high‐performance liquid chromatography (HPLC) fractionation of gangliosides without previous derivatization. These procedures make use of a reversed‐phase Lichrosorb RB‐8 or μBondapak RP‐18 column, and of a mixture of acetonitrile and 5 mM phosphate buffer, at fixed or varying volume ratios, as solvent system. Peak elution from the column is monitored by flow through reading of absorbance at 195 nm. Under all the described conditions HPLC is capable of resolving all common gangliosides and of separating each of them into four molecular species containing C18‐sphingosine, C18‐sphinganine, C20‐sphingosine, or C20‐sphinganine.

The analytical method has been successfully applied to fractionation of ganglioside mixtures from calf brain and to verification of homogeneity of singleganglioside preparations. It is suitable for quantitative purposes, with high sensitivity (detection limit, 0.1 nmole) and precision (SD less than 10% of mean values in the concentration range 0.1–50 nmoles). The semipreparative method, which provides successive cycles of analysis in a fully automated way, enables the preparation in 2–4 days of 100‐mg amounts of each molecular species starting from single gangliosides, like GM1 and GD1a. The preparative method makes use of acetonitrile‐phosphate buffer‐tetrahydrofuran as eluting solvent, and requires the addition to the starting ganglioside of the corresponding radioactive compound as tracer. This procedure, applied to GM1 ganglioside, is devised for processing up to 50 mg of ganglioside per analysis.
GM1 ganglioside facilitates the recovery of high‐affinity choline uptake in the cerebral cortex of rats with a lesion of the nucleus basalis magnocellularis
Authors: Pedata F. , Giovannelli L. , Pepeu G.
Published: January 2005

The effect of repeated administrations of GM1 monosialoganglioside on high‐affinity choline uptake (HACU) was investigated in the cerebral cortex of rats with a unilateral electrolytic lesion of the nucleus basalis magnocellularis. In saline‐treated rats, 4 days after lesion, a 38% and 14% decrease in HACU‐activity was found in the ipsilateral frontal and parietal cortex, respectively. A spontaneous recovery of HACU activity occurred within the next 20 days. In rats receiving daily injections of GM1 from the day of operation, no significant decrease in HACU activity was found in the lesioned hemisphere 4 days after the lesion. In contrast a 25% increase in HACU activity in the frontal area of the opposite hemisphere was detected. No effect of GM1 treatment could be seen on days 10 or 20 after lesion. The possibility that GM1 may exert its stimulatory effect on HACU independently of the well‐known effect of gangliosides on neuronal sprouting is discussed.
Gangliosides minimize behavioral deficits and enhance structural repair after brain injury
Authors: Sabel B. A. , Dunbar G. L. , Stein D. G.
Published: January 2005

Injections of GM1‐gangliosides (30 mg/kg, i. p.) in adult rats were shown to reduce behavioral deficits after brain lesions. This was observed (1) after bilateral electrolytic lesions of the caudate nucleus in a learning task involving negative reinforcement; (2) following aspiration lesions of the mediofrontal cortex in a learning task involving positive reinforcement; and (3) when rotational behavior was assessed after amphetamine or apomorphine injections in animals with partial hemitransections of the nigro‐striato‐nigral fibers. A detailed anatomical analysis of the latter study, using a retrograde tract‐tracing dye wheat germ agglutininhorseradish peroxidase (WGA‐HRP), provided evidence for ganglioside‐stimulated, neuronal reorganization of connections to the caudate nucleus.

Our findings support the notion that gangliosides reduce behavioral deficits following brain injury by preventing secondary neuronal degeneration and/or enhancing structural reorganization of remaining afferents, rather than by influencing denervation supersensitivity.
Effects of GM1 ganglioside on developing and mature serotonin and noradrenaline neurons lesioned by selective neurotoxins
Authors: Jonsson G. , Gorio A. , Hallman H. , Janigro D. , Kojima H. , Luthman J. , Zanoni R.
Published: January 2005

The effect of exogenous GM1 ganglioside on selective neurotoxin‐induced lesions of serotonin (5‐HT) and noradrenaline (NA) neurons in both the central and peripheral nervous systems has been investigated in developing and adult rats and mice by employing neuro‐ and histochemical techniques. 5,7‐Dihydroxytryptamine (5,7‐HT) was used to lesion 5‐HT neurons, and 6‐hydroxydopamine (6‐OH‐DA) was used to lesion NA neurons. In most lesion models investigated the neurotoxin causes primarily an axonal nerve terminal damage without notably affecting the perikarya. There was no evidence indicating that GM1 interferes with the primary and direct neurodegenerative actions of 5,7‐HT or 6‐OH‐DA on 5‐HT and NA nerve terminals, respectively. In all lesion models GM1 had in the chronic stage a counteracting effect on the neurotoxin‐induced nerve terminal lesion or enhanced regrowth. The present results are compatible with the view that GM1 has a regrowth‐stimulating effect and/or protective actions against secondary retrograde degeneration following the initial nerve terminal lesion induced by the neurotoxin.
Gangliosides' dual mode of action: A Working hypothesis
Authors: Janigro D. , Di Gregorio F. , Vyskocil F. , Gorio A.
Published: January 2005

Using in vitro preparations, we have tested the hypothesis that gangliosides, and more specifically GM1, may prevent progressive neural damage following a trauma by means of complex intracellular mechanisms that might be triggered originally by ganglioside interaction with neuronal membranes. We have recently shown that 2‐hr ganglioside incubation in vitro stimulates the membrane Na/K pump in neuromuscular preparations. However, 5–6‐hr incubation or in vivo treatment for 3 days with a daily injection of gangliosides at a dose of 1 or 10 mg/kg prevents the depolarization that normally occurs after several hours of exposure to K+‐free solutions. In such undepolarized muscles, the electrogenic Na+/K+ pump does not seem to be activated.

Hippocampal slices subjected to hypoxia undergo depolarization, which is reversed after oxygen readmission. The recovery phase is characterized by a huge hyperpolarization, probably reflecting electrogenic pump activity. In control preparations the depolarization occurs after 3.15 ± 0.4 min and has a value of 48.7 ± 5.7 mV; GM1 treatment for at least 4–5 hr increases the latency to 7.3 ± 2.3 min, and the depolarization is reduced to 31.8 ± 4.5 mV. This protective effect is accompanied by a reduced hyperpolarization in treated preparations. The ionic studies performed on neuromuscular preparations indicate that the protective effect may not be solely dependent on K+ leakage; however, the experiments are not conclusive and must be repeated with more direct methods.

The results obtained indicate a dual mode of action for gangliosides. The early one seems characterized by membrane‐enzyme activation, perhaps in relationship to their incorporation in the membrane, which could be compatible with previously described effects, such as enhancement of neuronal sprouting and neuritogenesis. The late one, occurring 4–5 hr after ganglioside addition in vitro, might reflect intracellular events and be compatible with the protective action exhibited by gangliosides against neural damage.
β‐Adrenergic and muscarinic cholinergic receptors: Overlap in their genetic determination
Authors: Nagy A. , Reilly M. , Lajtha A.
Published: January 2005

Significant positive correlation between the ligand binding values for β‐adrenergic receptors and those for muscarinic cholinergic receptors was found in five inbred strains of mice (r = 0.84) and in different vertebrate species (r = 0.99).

Comparative analysis of analogous receptor studies done on various brain regions in other laboratories revealed high positive correlation between regional binding values for these two receptors. In conjunction with the work presented here, an overlap in the genetic determination of β‐adrenergic and muscarinic cholinergic receptors is suggested.
Growth cone‐target interactions in the frog retinotectal pathway
Authors: Reh T.A. , Constantine‐Paton M.
Published: January 2005

The growth cones of retinal ganglion cell axons were studied in the optic tract and tectum with hoseradish peroxidase (HRP) histochemistry and electron microscopy. The ganglion cell growth cones has many morphological features similar to those described in vitro and in other in vivo systems. However, we found that some processes formed highly differentiated terminal arborizations, while retaining growth cones on many of their branches. In addition, ultrastructural examination of the tectal neuropil revealed that many ganglion cell axonal processes had characteristics of both growth cones and presynaptic endings. These findings are discussed in the context of the hypothesis of shifting connections and the evidence that retinotectal map formation involves several mechanisms, including a process that depends on the action potential activity in the optic fibers.
Cholinergic neurotransmission in the central nervous system of the snell dwarf mouse
Authors: Fuhrmann G. , Durkin T. , Thiriet G. , Kempf E. , Ebel A.
Published: January 2005

An unequal decrease in cholinergic activity has been evidenced in discrete brain areas in the growth hormone, thyroid‐stimulating hormone and prolactin deficient Snell dwarf mouse. The effect of the mutation's pituitary deficit on central cholinergic mechanisms appears to be selective: Normally high cholinergic activity areas such as striatum, olfactory tubercles, and hippocampus show strong alterations in this neurotransmitter system. Structures which appear earlier in ontogenesis are less affected, if at all. The lack of pituitary hormones seems to have effects on choline acetyltransferase activity and/or synthesis as well as on the development of high affinity (H. A.) cholinergic uptake mechanisms, both strongly defective in hippocampus and striatum. Therefore, a lower density of cholinergic terminals can be inferred. Furthermore, our observations are consistent with a close functional coupling of the choline H. A. transport and of subsequent choline acetylation. Acetylcholinesterase activity does not seem to be affected. Moreover, a compensatory effect at the postsynaptic level may have occurred due to developmental or functional plasticity for cholinergic responsiveness. In conclusion, the dwarf mouse seems to be a useful model for a better understanding of the influences of growth hormone and thyroid hormones on the development of central cholinergic mechanisms. It also provides the possibility to attempt a functional restoration of the deficient cholinergic neurotransmission and the behavioral disturbances which may be linked to them, by hormone replacement.
Modulation of anterior pituitary dopamine receptors by estradiol 17‐β: Dose‐response relationship
Authors: Ali S.F. , Peck E.J.
Published: January 2005

Exposure of ovariectomized rats to estradiol‐17‐β for 48–96 hr resulted in a dose‐dependent reduction in the number but not the Kd of D‐2 dopamine receptors of the anterior pituitary. No influence of estrogen was observed on dopamine or muscarinic acetylcholine receptors of caudate or hypothalamus. The dose‐response relationship observed suggested that the influence of estrogen is directly on the pituitary, not secondary to the alteration of dopaminergic systems in the hypothalamus.
False positiveness in a choline acetyltransferase assay caused by nonionic reducing reagents
Authors: Kobayashi Y. , de Vellis J.
Published: January 2005

Dithiothreitol and 2‐mercaptoethanol have been frequently used as protective agents of choline acetyltransferase (EC 2. 3. 1. 6) during its purification. We found that those reagents reacted with acetyl‐CoA to form products that were co‐extracted with acetylcholine into organic scintillation liquid to be erroneously detected as high enzyme activity. An anionic reducing reagent, such as sodium thioglycolate, does not cause the above problem and can be an alternative protective agent.
Nuclear uptake and retention of 3H‐dihydrotestosterone or one of its metabolites in the lower brainstem of the baboon
Authors: Sheridan P.J. , Weaker F.J.
Published: January 2005

The nuclear uptake and retention of androgen in the lower brainstem of the baboon was examined after the injection of 3H‐5α‐dihydrotestosterone. A specific topographical pattern of localization was observed in both motor systems and somatosensory systems. The pattern was similar to that reported in the rhesus monkey. The localization found in the primate differs from that of the rat in that the localization demonstrated in the rat is primarily restricted to motor systems.
Synthesis of myelin proteins and ultrastructural investigations in regenerating rat sciatic nerve
Authors: Alberghina M. , Bernocchi G. , Biggiogera M. , Viola M. , Karlsson J.O. , Manfredi‐Romanini M.G. , Giuffrida A.M.
Published: January 2005

Myelin protein synthesis, as well as ultrastructural and morphometric changes in regenerating peripheral nerve, was studied. Sciatic nerves of rats were crushed unilaterally; sham‐operated nerves of the contralateral side served as controls. For the in vivo experiments, rats were killed at selected periods after the nerves were crushed (30, 60, 90, and 120 days); seven days prior to killing, the animals were injected intravenously with L‐[4, 5−3H]leucine. For the in vitro experiments, proximal and distal segments of sciatic nerve and equivalent sham‐operated nerves were labeled with 3H‐amino acid mixture 90 days after axotomy. Purified myelin was isolated from nerve segments; specific radioactivity and gel electrophoretic patterns of proteins were analyzed. Cross‐sectional electron microscope (EM) preparations of proximal, distal, and contralateral segments of nerves also were examined. Results showed that the incorporation of labeled amino acids into total myelin proteins was enhanced significantly in the distal segment of sciatic nerves at all of the periods of regeneration studied. The yield of myelin protein per mm distal nerve segment increased as regeneration proceeded. The remyelination of fibers early after nerve crush was weak, whereas it gradually attained the normal range 90–120 days after axotomy. Morphometric analysis of myelin sheath thickness of regenerating axons was consistent with the data obtained for myelin protein synthesis.
Developmental expression of 2′,3′ ‐cyclic nucleotide 3′ ‐phosphohydrolase in dissociated fetal rat brain cultures and rat brain
Authors: Bansal R. , Pfeiffer S. E.
Published: January 2005

The development in primary dissociated rat brain cultures of 2′,3′ ‐cyclic nucleotide 3′ ‐phosphohydrolase (CNP) activity, the accumulation of CNP protein, and the number of cells accumulating this protein have been quantitatively determined as a function of time in culture. Parallel determinations have been made for the first two parameters for developing rat brain. The developmental profile of CNP enzymatic activity of CNP protein in culture paralleled that observed in rat brain, in which the period of most active development occurred 7–25 days after birth. Mean CNP activities of 5.6 and 8.1 μmol/min/mg total protein were recorded for the cultures and rat brain, respectively, at their maximal levels. The corresponding mean values for the CNP protein accumulation were calculated to be 138 and 150 pmol/mg total protein, respectively. Thus maximal specific activities of the CNP protein were estimated to be about 800 and 1,100 μmol/min/mg CNP protein for culture and rat brain enzyme, respectively. Approximately three million cells expressing CNP appeared in the cultures per dissociated fetal rat brain seeded. Each CNP + oligodendrocyte in culture had an average CNP activity of 3.2 pmol/min, and an average CNP protein content of 0.09 gmol (5.4 × 107 molecules), values which remained nearly constant during the course of development. Two pricipal conclusions are drawn from these data. First, the dissociated fetal brain culture system reproduces rather accurately the temporal developmental pattern of CNP expression occurring in the rat brain, but some important quantitativ differences occur which suggest the need for additional environmental stimuli missing in these cultures. Second, the quantitative increases in CNP specific activity and amount of CNP protein occurring during oligodendrocyte differentiation in these cultures are primarily the result of increases in the number of CNP + cells present which upon differentiation express very quickly, via an off‐on regulation, steady‐state levels of the enzyme.
Effects of tunicamycin on NGF binding and neurite outgrowth in PC12 cells
Authors: Baribault T. J. , Neet Kenneth E.
Published: January 2005

The effects of inhibition of glycosylation on nerve growth factor (NGF) binding and neurite outgrowth response of PC12 cells have been examined. Exposure to tunicamycin (1–10 μg/ml) for 24–36 hr eliminates the rapidly dissociating component of NGF binding and decreases the decreases the proportion of PC12 cells capable of elaborating neurits in a dose‐dependent manner. These decreased cellular responses are probably due to an underglycosylation of the NGF receptor, since the effects of tunicamycin are correlated with a decrease in 3H‐fucose incorporation rather than a general decline in cellular metabolism as measured by viability and protein syntheses. These results suggest that carbohydrate side chains are important for the function and/or orientation of the NGF receptor in PC12 cells and that the rapidly dissociating component of NGF binding may be associated with a minimum concentration of functional receptors per cell required for the full biologic response.
Excitatory effect of amino acids on identified neuron R14 of Aplysia. II. Neutral amino acids and structure‐activity relationships
Authors: Ichinose Mitsuyuki , McAdoo D. J.
Published: January 2005

The effects of 20 amino acids on identified neuron R14 of Aplysia kurodai were studid by conventional intracellular recording and voltage‐clamp techniques. Neutral α‐amino acids caused marked dose‐dependent depolarizations of the neuron. The effects of several amino acids applied simultaneously were additive. L‐isomers were much more effective than the corresponding D‐isomers. R14 was at most slightly depolarized by perfusion with 2‐aminoisobutyric acid and the non‐α‐amino acids β‐alanine and β‐ and γ‐aminobutyric acid. Putative neurotransmitters ACh, 5‐HT, dopamine, GABA, and taurine were much less effective in depolarizing R14 than neutral amino acids. Values of the Hill coefficient (nH) and the apparent dissociation constant (KA) were approximately 0.64 and 80 mM, respectively, for glycine. These results suggest that R14 has a general sensitivity to neutral amino acids and that amino acids in the hemolymph may be able to influence the electrical activity of R14.
Regulation of myelination: Axons not required for the biosynthesis of basal levels of the major myelin glycoprotein by schwann cells in denervated distal segments of the adult cat sciatic nerve
Authors: Poduslo J. F. , Berg C. T. , Ross S. M. , Spencer P. S.
Published: January 2005

The adult cat sciatic nerve was examined for Schwann cell biosynthesis of the major myelin glycoprotein (P0) in the distal segments after permanent nerve transection, where there is no axonal regeneration or myelin assembly. Endoneurial slices (intrafascicular tissue) from the distal segment of the desheathed cat sciatic nerves at 10 wk after transection and from normal adult desheathed brachial nerves were incubated with radioactive mannose; [3H]mannose incorporation into P0 was observed by fluorography after sodium dodecyl sulphate‐pore gradient electrophoresis (SDS‐PGE). Analysis of immune precipitates by SDS‐PGE after incubation of an aliquot of an endoneurial fraction with rabbit antichick P0 gamma globulin verified that the [3H]mannose‐labeled glycoprotein was P0. The level of incorporation of [3H]mannose into P0 and into other endoneurial glycoproteins in the normal brachial nerve from the adult cat was at substantially reduced levels compared with the transected nerve. Such incorporation was detectable by fluorography only after prolonged exposure to X‐ray film (15 days). As a result, the level of biosynthesis of P0 in the normal adult cat is substantially reduced, suggesting that the extent of active myelination in the adult cat nerve is at a low level. Furthermore, Schwann cells are capable of continued synthesis of P0 in the adult, permanently transected nerve in the absence of axonal influence, suggesting that axonal association is not an absolute requirement for specifying myelin protein synthesis.
Phosphorylative neuromodulation of the regulatory subunit of cyclic AMP‐dependent protein kinase type II in skeletal muscle
Authors: McLane J. A. , Squinto S. P. , Yeoh H. C. , Held I. R.
Published: January 2005

The phosphorylative neuromodulation of the regulatory subunit of protein kinase type II (R‐II) in cytosolic fractions from denervated and sham‐operated, contralateral soleus muscles of the rat was evaluated. The denervation‐induced increase in the 32P‐phosphorylation of R‐II is not related to an increased dephosphorylation by cation‐dependent or cation‐independent protein phosphatases in the cytosolic fractions. The level of 32P‐phosphorylation of an exogenous heptapeptide substrate (Kemptide) by dissociated catalytic subunits of cyclic AMP‐dependent protein kinase in cytosolic fractions from denervated and sham‐operated solei did not differ. Also, no change in the concentration of cytosolic R‐II assessed by competitive enzyme‐linked immunosorbent assays (ELISA) was found after denervation. However, the in vitro 32P‐phosphorylation of R‐II in these samples was increased. Taken together, our results suggest that the increased availability of autophosphorylatable sites reflects an in vivo modulation of R‐II phosphorylation rather than a significant change in total R‐II content.
Altered respiratory response to substance P in capsaicin‐treated rats
Authors: Towle A. C. , Mueller R. A. , Breese G. R. , Lauder J.
Published: January 2005

The present investigation sought to examine the importance of substance P in the altered respiratory activity after neonatal capsaicin administration. Halothaneanesthetized adult rats given capsaicin neonatally exhibit a decreased basal minute ventilation with PaCO2 equal to and PaO2 greater than vehicle injected controls. In addition, the minute ventilation‐PaCO2 curve was displaced to the right. Acute bilateral cervical vagotomy severely blunted the minute ventilation response to PaCO2 and abolished the differences in ventilation between capsaicin treated and control rats. Neonatal capsaicin significantly reduced pons‐medulla substance P content but not TRH, serotonin or 5‐hydroxyindole acetic acid. Immunohistochemical studies revealed that substance P fibers of the trigeminal spinal nucleus were the most severely affected in the brain stem and that substance P fibers in the lung were totally absent.

The intracerebroventricular administration of substance P increased minute ventilation similarly in both control and capsaicin treated rats, largely as a result of increases in tidal volume. The minute ventilation‐PaCO2 curve was similar in both groups after substance P administration. Simultaneous administration of the peptidase inhibitor captopril with substance P increased the respiratory response to substance P in normal rats. Administration of captopril to capsaicin treated rats restored the ventilation‐PaCO2 curve to the position observed in normal rats. The hypotensive response to intracerebroventricular captopril alone in control rats was less profound in rats given neonatal capsaicin.

These results are consistent with the thesis that respiratory depression after capsaicin treatment is at least in part due to the loss of substance P primary afferent nerve terminals in the brain stem, suggesting that substance P fibers in the brain stem may participate in the normal modulation of respiratory activity.
Partial characterization of a brain extract factor(s) inhibitory to transformed neural cells
Authors: Schengrund C.‐L. , Repman M. A.
Published: January 2005

The factor(s) present in extracts prepared from the brains of newborn A/J or C57B1/6 mice, with inhibits S20Y neuroblastoma cell growth in vitro, was partially characterized. Twice as much ingibitory activity was extracted per gram wet weight of brain than torso, and inhibitor recovery was greatest in extracts prepared from brains of mice 1 week or less in age. The inhibitory factor(s) was water‐soluble and was stable to heating at 100°C, to freezing, and to lyophilization. It was susceptible to the action of pronase. The factor(s) behaved like a molecule of molecular weight ˜ 700 upon passage through ultrafiltration membranes. Growth of rat hepatoma (H4), murine melanoma (B16), and transformed murine fibroblasts (WT19 and B6‐HCMV) was not significantly inhibited by brain extract. Growth of rat glioma cells (C6) was significantly reduced but to a lesser degree than that of murine neuroblastoma cells (S20Y and N 115) and glioma cells (G26‐20). These results suggest that the inhibitor expresses a cell specificity.
Purification of L‐Glutamate decarboxylase from rabbit brain and preparation of a monospecific antiserum
Authors: Brandon C.
Published: January 2005

L‐Glutamate decarboxylase (GAD), the enzyme responsible for the biosynthesis of gamma‐aminobutyric acid, has been purified from rabbit brain and used for the production of a monospecific rabbit antiserum. The enzyme was purified approximately 500‐fold by a combination of ion exchange, hydrophobic, hydroxyapatite, and gel filtration column chromatography, and preparative polyacrylamide gel electrophoresis. The preparation thus obtained was injected into a rabbit, producing a polyspecific antiserum. This first antiserum was used for the preparation of an immunoprecipitate containing GAD; injections of the immunoprecipitate into a second rabbit led to the production of a second, monospecific anti‐GAD serum. The specificity of the antiserum for GAD has been demonstrated by enzyme precipitation, immunodiffusion, line immunoelectrophoresis, gel filtration chromatography of GAD/anti‐GAD immune complexes, and immunocytochemistry in the rat cerebellum.
Binding sites for glycosaminoglycans on developing sympathetic neurones
Authors: Vidovic M. , Hill C. E. , Hendry I. A. , Parish C. R.
Published: January 2005

Recent studies have suggested that cell to cell communication in the immune system is mediated by cell surface receptors for glycosaminoglycans (GAGs) [Parish et al, 1984]. The intention of this study was to see whether similar recognition molecules for GAGs are present on sympathetic neurones. A mechanical dissociation technique was used to isolate neurones from superior cervical ganglia (SCG) of rats aged between gestational day 19 and postnatal day 21. Receptors for GAGs on sympathetic neurones were detected by the ability of neurones to form rosettes with sheep red blood cells coupled with one of 12 different GAGs. It was found that SCG cells bind to all the GAGs tested. In addition, a range of developmental binding patterns for the various GAGs was found.
Chick embryo myotubes contain transferrin receptors and internalize and recycle transferrin
Authors: Stamatos C. , Fine R. E.
Published: January 2005

Embryonic chick skeletal myotubes grown in cell culture require transferrin to provide iron for proliferation and differentiation. We demonstrate here that cultured myotubes contain transferrin receptors as demonstrated by the finding of specific, saturable, and reversible high‐affinity binding sites. Scatchard analysis of equilibrium binding data indicates an apparent Kd of 37 nM and one muscle cell equivalent contains 7,500 transferrin receptors. Myotubes exhibit a Kd 100 times higher for apotransferrin than for iron‐saturated transferrin.

Internalization of specifically bound transferrin is temperature dependent and occurs rapidly at 37°C with a steady state reached after 10 min. Internalization studies using either 125I‐ovotransferrin or 55Fe‐ovotransferrin suggest that transferrin is internalized, depleted of iron, and recycled intact to the extracellular medium as shown in other cell systems. Autoradiography of muscle cell cultures incubated with 125I‐ovotransferrin at 4°C reveals clusters of receptors along the myotubes.

The possible mechanisms by which transferrin is supplied to muscle in vivo are discussed in light of the evidence that motor neurons contain transferrin.
A biomolecular approach to the study of the expression of specific genes in the retina
Authors: McGinnis J.F. , Leveille P.J.
Published: January 2005

The central nervous system contains a number of populations of neurons that are morphologically and functionally distinct. To study the genes responsible for the development and maintenance of proteins with unique structural and biochemical properties, polyspecific antisera were produced against normal mouse retinal proteins and used in combination with the rd (retinal degeneration) neurological mutant of the mouse to immunologically identify specific retinal proteins. Western transfer analysis of the proteins present in normal and in mutant retinas identified three classes of neural proteins: (1) those found only in normal retina; (2) those found in normal and in photoreceptorless retinas but not in other tissues; and (3) those found in both normal and mutant retinas, as well as in brain, but not elsewhere. Some of these class 1 proteins were shown to be present in the retinas of other species, including humans, suggesting their importance in the process of vision. The poly A + RNA was isolated from the retinas of normal mice and used to generate a cDNA expression library in lambda gt‐11. This library was screened with polyspecific antisera absorbed with the proteins present in mutant retina, and a number of immunologically positive plaques were cloned. Four of these were shown to code for rhodopsin, the major visual protein in mammalian retinas. The approach described is applicable to other systems in order to generate specific immunological and recombinant DNA probes for examining the expression of specific genes during development and differentiation.
Chronic antidepressant treatment and mouse brain 3H‐imipramine binding
Authors: Severson J.A. , Anderson B.
Published: January 2005

Chronic pretreatment of mice with the monoamine oxidase type B inhibitor (–) deprenly resulted in an increase in the density of cerebral cortical 3H‐imipramine binding sites and a decrease in the density of cerebral cortical beta‐adrenergic receptors. In contrast, pretreatment of mice with the tricyclie antidepresents imipramine and desipramine did not alter the density of cerebral cortical 3‐Himipramine binding sites. Imipramine and desipramine treatment decreased the density of beta‐adrenergic receptors. Haloperidol pretreatment resulted in an increase in the density of striatal D‐2 dopamine receptors, but did not alter the density of cerebral cortical 3H‐imipramine binding sites or beta‐adrenergic receptors. These data suggest that brain 3H‐imipramine binding sites can be regulated by pharmacological pretreatment, but that this regulation may not occur for all antidepresents.
Immunocytochemical localization of nerve growth factor in mouse kidney
Authors: Salido E.C. , Barajas L. , Lechago J. , Laborde N.P. , Fisher D.A.
Published: January 2005

The presence of NGF in mouse kidney was investigated using immunocytochemical methods. Female and male adult Swiss‐Webster mouse kidneys were fixed by perfusion with 4% paraformaldehyde or Zamboni's fixative. The kidneys were forzen, and serial sections were prepared. Rabbit NGF antiserum was used for the primary incubation, and the avidin‐biotin complex immunoperoxidase procedure was utilized for immunostaining. NGF immunorectivity was localized in the apical and perinuclear cytoplasm of cells linig the late is distal nephorn, in a portion that corresponds to the connecting tubule. This portion of the nephron has been consistently observed in close anatomical to relationship with arterioles, which are known to be richly innervated. Some cells of the connecting tubule, correspondind to intercalated cells, lacked NGF immunoreactivity.