bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate effects on the dynamics of neurite growth in vitro: A quantitative multi‐parametric analysis
Authors: Saxod Raymond , Bizet Marie‐Claude
Published: April 2003

Embryonic chick dorsal root ganglia were cultured in serum‐free medium on natural (collagen, fibronectin and hyaluronic acid) and artificial (polylysine and polyornithine) substrata. The movement of individual growth cones was quantified by measuring five parameters using time‐lapse cinematography combined with a digitizing‐computer system, and the neurite behaviour was compared between the different substrata with multivariate statistical methods. For each substratum, the morphometry of the growth cone was quantified by measuring six morphological parameters. The most discriminative parameters proved to be mean velocity and straightness index for neurite extension, and projected area and cumulated length of filopodia for growth cone morphometry. A good correlation was obtained between behavioural and morphological parameters and the larger the cone area and the filopodia length, the faster and the straighter the neuritic growth. Both quantitative analyses showed highest values for polyornithine and the lowest for hyaluronic acid, and divided the substrata into two opposite groups, artificial and natural. It is concluded that growth cone behaviour and conformation is modulated by substratum properties.
The effect of exogenous human ras and myc oncogenes in morphological differentiation of the rat pheochromocytoma pc12 cells
Authors: Spandidos Demetrios A.
Published: April 2003

An electroporation technique was employed to study the effect of oncogenes H‐ras and c‐myc after their short‐term expression in the rat pheochromocytoma PC12 cells. It was found that within 6 days after electroporation the mutant T24 H‐ras 1 gene induced differentiation of PC12 cells whereas the c‐myc blocked NGF‐induced differentiation. The induction of differentiation by the T24 H‐ras gene may suggest a physiological role of the ras gene in cell differentiation as well as in cell proliferation.
Correlated ultrastructural damage between cerebellum cells after early anticonvulsant treatment in mice
Authors: Fishman Rachelle H. B. , Ornoy Asher , Yanai Joseph
Published: April 2003

The anticonvulsants phenobarbital and diphenylhydantoin administered early in life to mice resulted in significant and long‐lasting ultrastructural damage, including abnormalities of mitochondria, myelin sheaths and lamellar inclusion bodies inside identified cells throughout the cortical layers of the cerebellum in treated vs control mice. The magnitude, distribution and duration of damage was age and treatment specific. No differences were detected in density of parallel fiber processes nor in synapse density within the molecular layer. Neuron profiles containing damaged organelles were not homogeneously distributed but made up only a small fraction of the total cell population examined. In our experiments, there was an overall within‐animal correlation explaining 45% of the magnitude of damage in different cerebellar regions, but between synaptically connected cells, specifically mossy fiber axon varicosities and granule cell dendrite profiles, the subset population ratio of damaged‐to‐total mitochondria was highly significantly correlated (70–90%; P<0.001). We hypothesized that some correlated transneuronal degeneration and death in the central nervous system may have a transynaptically regulated component that first appears as correlated damage between synaptically connected cells, perhaps regardless of the degree of toxicity. The orderly cytoarchitecture and cell connections of the cerebellar cortex can be used to study these patterns of degeneration.
Development of homospecific activity of GABA‐transaminase in the mouse cerebral cortex and cerebellum and in neurons cultured from these brain areas
Authors: Schousboe Inger , Larsson Orla M. , Schousboe Arne
Published: April 2003

The homospecific activity of GABA‐transaminase (EC 2.6.1.19; GABA‐T) in brain or neurons was determined as a function of development in vivo or in culture by measuring the enzyme activity together with the relative amount of GABA‐T apoenzyme by the aid of a monospecific anti‐GABA‐T antibody. It was observed that both in cerebral cortex and cerebellum in vivo and in neurons cultured from these brain regions the homospecific activity of GABA‐T changed during development. By incubation of tissue extracts with similar extracts in which GABA‐T activity had been selectively and irreversibly destroyed with gamma‐vinyl GABA (Vigabatrin) it was established that this change in homospecific activity was at least partly due to the presence of an endogenous activator of GABA‐T. The results point towards a rather complex endogenous regulation of GABA‐T during development in vivo and in vitro.
Developing rat cerebellum—II. Effects of abnormal thyroid states and undernutrition on hyaluronic acid
Authors: Normand G. , Vitiello F. , Clos J. , Gombos G.
Published: April 2003

The early postnatal pattern of hyaluronic acid (HA) deposition in rat cerebellum is affected by thyroid deficiency, thyroxine treatment and undernutrition. The modification of HA ontogenesis apparently reflects the smaller number of cells formed in undernourished rats, or alterations of cell maturation (accelerated in thyroxine‐treated and slowed down in thyroid‐deficient rats). The developmentally regulated:loss of tissue water is also affected in the three conditions; this can be correlated with the roughly simultaneous disappearance of extracellular, but not of total, HA.
Vimentin‐GFAP transition in primary dissociated cultures of rat embryo spinal cord
Authors: Bignami Amico , Dahl Doris
Published: April 2003

Primary dissociated cultures derived from 15‐day‐old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria® and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin‐positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4–5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP‐positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin‐positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.
Growth promoting effects of IGF‐I on fetal hypothalamic cell lines under serum‐free culture conditions
Authors: Torres‐Aleman Ignacio , Naftolin Frederick , Robbins Richard J.
Published: April 2003

Recent evidence indicates that the insulin‐like family of peptides may act as endogenous trophic factors in the central nervous system. To further examine this possibility we have investigated the effects of three insulin‐like peptides on thein vitro growth of fetal hypothalamic cell lines. Two virally transformed rat hypothalamic cell lines which have been developed in our laboratory (A‐6 and F‐12) were used. Cells were plated at varying densities and cultured in the presence or absence of either insulin‐like growth factor I (IGF‐I), insulin, or multiplication stimulating activity (MSA or IGF‐II), in serum‐free medium for 1 wk. Cell growth was assessed by counting or by measuring cellular incorporation of3H‐thymidine. Of the three peptides tested IGF‐I was the most potent in eliciting cell growth. Insulin also stimulated growth of both cell lines, but was 100 times less potent for A‐6 cells while it was equipotent with IGF‐I in F‐12 cells. MSA had no effect on either cell line. Both IGF‐I and insulin showed dose‐response effects in increasing cell growth. We also found that the two cell lines had the greatest response to IGF‐I at low cell densities. Finally, time‐course experiments suggested that a continued presence of the peptide is essential for the growth‐promoting effects. We conclude that IGF‐I is a potent growth factor for virally transformed cell lines derived from the rat fetal hypothalamus. Since both IGF‐I immunoreactivity and IGF‐I receptors have been located in this diencephalic area these results suggest that IGF‐I may constitute a mitogenic signal for hypothalamic cells during neurogenesis.
Retinoids increase perinatal spinal cord neuronal survival and astroglial differentiation
Authors: Wuarin L. , Sidell N. , de Vellis J.
Published: April 2003

In this report we demonstrate that retinol and retinoic acid (RA) increase the survival and morphological differentiation of rat spinal cord neurons in vitro. Micromolar amounts of retinol and RA increased the number of surviving neurons by 2‐ to 3‐fold and affected neuritic density resulting in increased secondary and tertiary processes compared to untreated sister cultures. A marked morphological differentiation of the astrocyte population in conjunction with an antiproliferative effect in the presence of retinoids were apparent. These trophic effects occurred mainly after 5 days in vitro, a time that corresponds to the time of birth in vivo. Retinoic acid exerted a direct trophic effect on spinal cord neurons in the absence of glial cells while retinol lost its effectiveness. Metabolic labeling suggested that retinol is converted to the biologically active RA within astrocytes but not in neurons. Taken together, our results have demonstrated direct trophic effects of RA on spinal cord neurons and have suggested another role for astrocytes in the maintenance of normal neural physiology by regulating RA concentrations through the oxidation of retinol.
Changes in the expression of acetylcholinesterase molecular forms during rat heart development
Authors: Nyquist‐Battie Cynthia
Published: April 2003

The development of the molecular forms of acetylcholinesterase was studied in rat heart during the perinatal period. In this study, the activity of acetylcholinesterase increased both per unit wet weight and per unit protein from post‐conception day 14 to day 42. Additionally, the activity of atrial acetylcholinesterase per unit wet weight increased more rapidly after birth than that of ventricular acetylcholinesterase. The percent contribution of the various molecular forms to the total acetylcholinesterase pool in heart changed dramatically from the fetal to the neonatal period. This switch primarily consisted of a decrease in the ratio of the asymmetric to globular forms. Thus, the specific activity of globular forms increased while that of the asymmetric forms remained relatively stable. When the atria and ventricles were examined separately at 19 days post‐conception, the percent contribution of the individual molecular forms in the two cardiac areas was different. The atria contained a pool of acetylcholinesterase forms similar to postnatal heart while the ventricles contained a pool of acetylcholinesterase forms with a lower globular to asymmetric ratio. Finally, this study showed that greater than half of the acetylcholinesterase pool was inhibited by incubating hearts from fetal rats with echothiopate iodide, suggesting that a large portion of acetylcholinesterase catalytic sites are externalized in fetal heart.
Developmental profiles of cholinergic activity in the habenulae and interpeduncular nucleus of the rat
Authors: Contestabile A. , Virgili M. , Barnabei O.
Published: April 2003

Choline acetyltransferase (ChAT) was measured in the habenula and in the interpeduncular nucleus of rats from 1 to 12 weeks of age. A remarkable degree of parallelism was shown by the developmental curves in the two nuclei. In both cases the highest level of enzyme activity was reached at 3 weeks of age and was followed by some decrease towards adult values. A statistically highly significant correlation was demonstrated between ChAT levels in the two nuclei at the various developmental stages. The rise of the cholinergic marker was slightly advanced in the habenula in comparison with the interpeduncular nucleus. The present data may be useful for studies focused on neonatal synaptogenesis, plasticity and synaptic neurochemistry of this relatively simple model of brain connections.
Accelerated neuromuscular development in the rat with prenatal exposure to nicotine
Authors: Rose Kenneth J. , Strand Fleur L.
Published: April 2003

Nicotine, administered to pregnant Sprague‐Dawley rats either throughout gestation or during two selective prenatal periods before or after muscular innervation (i.e. before or after gestational day 13), accelerated the maturation of developing nerve and muscle as viewed in 2‐week‐old offspring. Prenatal treatment of rats with nicotine (tartrate salt; 0.25 mg/kg twice a day; i.p.) induced marked changes in the contractile force and speed of the developing extensor digitorum longus (EDL) muscle when tested in situ on 14 to 15‐day‐old pups. In all nicotine treatment groups isometric twitch and tetanus half contraction durations were shorter than controls. Use of 2,3,5‐triphenyltetrazolium chloride, a biochemical indicator for cellular respiration, showed that totigestational nicotine administration increases cellular metabolic activity of EDL muscle. Hypertrophy of adrenal glands in these animals also was noted. While each of the three courses of nicotine administration accelerated muscle development and maturation, nicotine during the first half of prenatal development, prior to EDL muscle innervation, produced a more dramatic effect than nicotine during the second half of prenatal life.
Multi‐determinate regulation of neuronal survival: Neuropeptides, excitatory amino acids and bioelectric activity
Authors: Brenneman Douglas E. , Chang Yu , Nelson Phillip G.
Published: April 2003

Neuronal survival of dorsal root ganglion‐spinal cord cultures was determined after treatment with vasoactive intestinal peptide (VIP) and an antagonist to the N‐methyl‐d‐aspartate receptor (NMDA). Blockade of NMDA receptors with 2‐amino 5‐phosphonovaleric acid (AP5) produced a bi‐phasic response on neuronal survival: low concentrations (0.1 μM) resulting in greater survival and higher concentrations (100 μM) causing cell death. VIP, a substance with demonstrated neurotrophic properties in vitro, prevented the neuronal cell death associated with high concentrations of AP5, while having no additive effect on the survival‐promoting action of low levels of AP5. Electrophysiological studies indicated that APS, although reducing high frequency bursting activity, did not significantly reduce the abundant on‐going asynchronous activity present in these cultures of high density neuronal networks. These data indicate that excitatory amino acids have more than one action that can influence neuronal survival during development and that VIP can increase neuronal survival in bioelectrically active cultures when NMDA channels are blocked. Together with previous studies, these data suggest that multiple neurochemical inputs serve to determine the survival of spinal cord neurons during development, perhaps through one final common pathway: intracellular calcium regulation.
Oxidative mechanisms underlying methyl mercury neurotoxicity
Authors: Sarafian T. , Verity M. Anthony
Published: April 2003

Cerebellar granule cells from 5–12‐day‐old rats can be incubated in suspension at 37°C for up to 3 hr with minimal decline in viability. Methyl mercury was found to produce time‐ and concentrationdependent cell killing with >85% cell death after 3 hr exposure to a concentration of 20 μM. Previously characterized inhibition of protein and RNA synthesis as well as known methyl mercury‐induced defects in cellular ATP production have been shown to be incapable of causing this degree of cell death. Here we report that methyl mercury induced a concentration‐dependent increase in membrane lipoperoxidation and a rapid decline in reduced glutathione in this cerebellar neuronal preparation. Hydrogen peroxide at 5 mM was found to closely reproduce each of the cytotoxic effects manifested by methyl mercury suggesting that oxidizing conditions produced by methyl mercury may account for the observed cell death. Methyl mercury‐induced lipoperoxidation was not the cause of cell death since malonaldehyde production could be blocked by α‐tocopherol or EDTA without appreciably protecting against cell death. Significant protection from methyl mercury‐induced cell death was observed, with EGTA, deferoxamine and KCN. We propose that oxidative events contribute to the toxic mechanism of action of methyl mercury in isolated cerebellar granule neurons.
Effect of vitamin E succinate and a cAMP‐Stimulating agent on the expression of c‐myc, N‐myc and H‐ras in murine neuroblastoma cells
Authors: Cohrs Randall J. , Torelli Sylvia , Prasad Kedar N. , Edwards‐Prasad Judith , Sharma Opendra K.
Published: April 2003

d‐Alpha‐tocopheryl succinate (vitamin E succinate) at a concentration of 11.3 μM inhibited growth and reduced the expression of c‐myc, N‐myc and H‐ras specific mRNAs in murine neuroblastoma cells (NBP2) in culture. R020‐1724 [4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone], an inhibitor of cyclic AMP phosphodiesterase, also inhibited growth and reduced the expression of these oncogenes. Vitamin E succinate treatment caused the formation of two c‐myc related transcripts of 1.9 and 3.7 kb; however, R020‐1724 treatment did not. These results suggest that the inhibition of growth is sufficient to reduce the expression of c‐myc, N‐myc and H‐ras in NB cells in culture, but it is not sufficient to produce two c‐myc related transcripts.
Myelination and glial ensheathment of purkinje cells in cerebellar cultures are not inhibited by antibodies to the neural cell adhesion molecule, N‐CAM
Authors: Seil Fredrick J. , Herndon Robert M.
Published: April 2003

Mouse cerebellar cultures were exposed to anti‐N‐CAM antibodies throughout their in vitro development. Some cultures were stripped of myelinating oligodendrocytes and functionally competent astrocytes by treatment with cytosine arabinoside (Ara C), while others were left untreated and were potentially capable of forming myelin around axons and astrocytic sheaths around Purkinje cell somata and dendrites. As expected, the antibodies inhibited axonal fasciculation in the Ara C treated cultures. However, the same antibodies had no discernible effect on formation of myelin or astrocytic sheaths in cultures not treated with Ara C. N‐CAM is expressed on the surfaces of neurons, oligodendroglia and astrocytes, and has been proposed as the signal molecule governing both kinds of neuron‐glia interactions. The observations of the present study strongly suggest, however, that N‐CAM does not have an indispensable role in such interactions.
Parallel expression of synaptophysin and evoked neurotransmitter release during development of cultured neurons
Authors: Ehrhart‐Bornstein Monika , Treiman Marek , Hansen Gert H. , Schousboe Arne , Thorn Niels A. , Frandsen Aase
Published: April 2003

Primary cultures of GABAergic cerebral cortex neurons and glutamatergic cerebellar granule cells were used to study the expression of synaptophysin, a synaptic vesicle marker protein, along with the ability of each cell type to release neurotransmitter upon stimulation. The synaptophysin expression and neurotransmitter release were measured in each of the culture types as a function of development for up to 8 days in vitro, using the same batch of cells for both sets of measurements to obtain optimal comparisons. The content and the distribution of synaptophysin in the developing cells were assessed by quantitative immunoblotting and light microscope immunocytochemistry, respectively. In both cell types, a close parallelism was found between the temporal pattern of development in synaptophysin expression and neurotransmitter release. This temporal pattern differed between the two types of neurons. The cerebral cortex neurons showed a biphasic time course of increase in synaptophysin content, paralleled by a biphasic pattern of development in their ability to release [3H]GABA in response to depolarization by glutamate or elevated K+ concentrations. In contrast, a monophasic, approximately linear increase in the synaptophysin content and stimulated [3H]D‐aspartate release was found in the cerebellar granule cells. These results, particularly regarding the GABAergic neurons, offer correlative evidence in support of the notion that a vesicular pool of these amino acid neurotransmitters may be intimately involved in their release, subsequent to depolarization stimuli.
Chronic blockade of bioelectric activity in neonatal rat cortex grown in vitro: Morphological effects
Authors: Ruijter J.M. , Baker R.E. , De Jong B.M. , Romijn H.J.
Published: April 2003

Culture thickness, numerical density of neurons and neuronal survival were studied in timed series of control and tetrodotoxin‐silenced neocortical cultures to provide information on the role of bioelectric activity on neuronal development. In control cultures, culture thickness and number of surviving neurons decrease during the first weeks in vitro, but remain constant between 2 and 3 weeks indicating that the cultures are essentially mature. In the 4th week in vitro a further decrease in surviving neurons was observed. In tetrodotoxin‐treated cultures the number of surviving neurons decreased significantly between 1 and 2 weeks in vitro, to remain constant thereafter. However, culture thickness significantly increased at 3 and 4 weeks in vitro after an initial drop between 1 and 2 weeks. Compared to age‐matched controls at 2 and 3 weeks in vitro, only ca 50% of the neurons survived the loss of bioelectric activity. Similar differences were present between 1 and 2 weeks. Thus, the loss of all measurable bioelectric activity induces neuronal death in neocortical explants, but promotes neuropil formation by the surviving cells.
Extrafusal and intrafusal motor units in the kitten
Authors: Gregory J.E. , Proske U.
Published: April 2003

In the lateral flexor digitorum longus muscle (FDL) of anaesthetized kittens aged 3–21 days, alpha (α) and gamma (γ) motoneurone conduction velocities were measured together with motor unit tensions. Conduction velocity lay in the range 8–27 msec−1 for α motoneurones and 2–10 msec−1 for γ motoneurones. Motor unit tetanic tensions were 3–47 mN with the largest units being 2–2.2% of whole muscle tension. The hypothesis was tested that motor axons conducting within the γ range and presumed to be innervating the intrafusal fibres of muscle spindles, on stimulation, produced measurable tension. Stimulating γ motoneurones in the adult produces no tension. The hypothesis was based on histological observations that while in the adult, intrafusal fibres were about one third of the size of extrafusal fibres, in kittens the two were nearly equal, both in length and diameter. It was shown by means of signal averaging during stimulation of ventral root filaments that whenever tension was recorded in the muscle an impulse could be detected in the muscle nerve conducting at α motoneurone tempo. It is concluded that in the kitten, despite the fact that intrafusal and extrafusal fibres are of similar size, stimulating single γ motoneurones does not develop measurable tension.
Activation of muscarinic cholinergic receptors stimulates inositol phosphates synthesis in the developing avian cochlear duct
Authors: Bartolami Sylvain , Mayat Ebrahim , Lippe William R. , Rebillard Guy , Pujol Rémy
Published: April 2003

We previously reported that the inositol phosphates (IPs) synthesis is induced by muscarinic agonists in the rat cochlea and that this stimulation is maximal at postnatal day 12. This peak response is concomitant with the onset of the efferent synaptogenesis at the outer hair cell level. Whether the correlation between this neuronal plasticity and the enhanced IPs formation is unique to the rat or a general feature of the developing vertebrate cochlea is not known. To examine this question, we measured, in the presence of LiCl, the accumulation of (3H)‐IPs induced by carbachol, in the developing chick cochlear duct during a period ranging from embryonic day (E) 8 to post‐hatching day (P) 20. Carbachol (1 mM) causes a significant increase of IPs formation relative to basal values at all ages. This IPs accumulation is maximal at E8 (1854% of the basal level), then, rapidly decreases until P13 when it reaches a steady‐state level of 294% of the basal level. Strikingly, this gradual decline in IPs formation is interrupted between E15 and E19, by a transient increase in IPs synthesis. This rise peaks at E16 with a stimulation value of 757% of the control level. This maximal stimulation is inhibited by atropine in a dose‐dependent manner, as is the case at E9, suggesting the involvement of muscarinic receptors. Interestingly, the occurrence of the peak response is concomitant with the plastic events associated with the maturation of the efferent innervation of the cochlear duct. Thus, these results suggest that there may be a correlation between cochlear plasticity and enhanced IPs synthesis, which is not species‐specific. The possible significance of the overall decrease in IPs formation, occurring during embryonic development, is discussed.
Different degradation rates of junctional and extrajunctional acetylcholine receptors of human muscle cultured in monolayer and innervated by fetal rat spinal cord neurons
Authors: Braun Serge , Askanas Valerie , Engel W. King
Published: April 2003

It is well demonstrated that in intact animals the degradation rate of the junctional acetylcholine receptor (AChR) is significantly slower than that of the extrajunctional receptor. Such data, however, are not available for human AChRs because the required experimentation cannot be performed in humans. We have now studied the degradation rate of the junctional and extrajunctional AChRs, utilizing our tissue culture model, in which well‐differentiated neuromuscular junctions (NMJs) form on human muscle cultured in monolayer and innervated long‐term by fetal rat spinal cord neurons. Half‐life of AChRs was studied by a method utilizing the autoradiography of 125I‐αbungarotoxin and computerized video image analysis. Extrajunctional AChRs degraded with a half‐life of 1.3 days whereas junctional AChRs degraded with a half‐life of 3.5 days. Our studies demonstrate for the first time that in innervated cultured human muscle: (a) the life span of human junctional AChR, is approximately 3 times longer than that of the extrajunctional AChR and (b) the stability of human AChR is neuronally regulated. This system can now be applied to evaluate the influence of pharmacologie agents on the stability of human junctional AChR, which is of potential importance in the treatment of myasthenia gravis and other diseases of the NMJ.
Modifications of synaptosomal plasma membrane protein composition in various brain regions during aging
Authors: Ragusa Nicolo' , Villa Roberto F. , Magri' Grazia , Ingrao Francesca , Gorini Antonella , Turpeenoja Leena , Stella A.M. Giuffrida
Published: April 2003

The age‐dependent modifications of synaptosomal plasma membrane protein composition in three different rat brain regions (cerebral cortex, cerebellum and striatum) at various ages (4,12 and 24 months) were studied. The proteins were separated by gel‐electrophoresis and the quantity of the different polypeptides was determined densitometrically from the stained gels.

In the three brain regions examined several age‐related modifications in the amount of the synaptosomal plasma membrane proteins were observed. In particular a significant decrease in the content of some synaptosomal plasma membrane proteins at 24 months of age was found. The agerelated modifications in the protein composition of synaptosomal plasma membrane may cause changes in many brain functions, such as neurotransmission, ionic transport and enzyme activities.

Particularly interesting is the decrease of a protein with 18 kDa mol. wt. This protein has been identified as calmodulin by immunoblotting assay. The decrease in the amount of this protein may be correlated to the impairment of several Ca2+‐ requiring processes in the aging brain.
Development of membrane currents in mammalian neuroventricular cells from the early neural tube stage in vitro: Aspects of the neuronal lineage
Authors: Wiedehage Johannes , Schmidtmayer Johann , Buse Eberhard
Published: April 2003

Neuroepithelial cells from the murine brain anlage at the early neural tube stage (embryonic day 9½, stage 15) were cultured. Their morphology and the development of membrane currents were studied during 2 weeks in culture. Immediately after dissociation the cells were equal in shape and no morphological or ultrastructural differences were evident. Patch‐clamp measurements within the first 2 h, however, showed different membrane properties. Either sodium inward currents or potassium outward currents were observed in these undifferentiated cells, but no combined inward and outward currents were found. This would mean that some of the neuroventricular cells, but obviously not all of them, display neuronal membrane properties (sodium currents) in the immediate post neural plate stage. After 1 day, developing neurons could be identified morphologically by neurotubuli and process formation. The transformation of these cells into neurons was electrophysiologically characterized by an increasing sodium channel density and the expression of various kinds of potassium channels. After 4 days the vast majority of the neurons was electrically excitable, i.e. they could generate action potentials. The standard electrical profile at this time was characterized by a sodium inward current followed by a delayed potassium outward current. In the following days the complexity of membrane currents increased in some neurons by the emergence of transient potassium currents. After 2 weeks in culture different neuronal phenotypes could be identified.
Morphometric and time lapse analyses of rapid‐onset neurites stimulated by cycloheximide in NG108‐15 cells
Authors: Smalheiser Neil , Rossulek Michelle
Published: April 2003

We previously reported that the laminin‐induced outgrowth of neurites in the first 4 hr after plating (‘rapid‐onset neurites’) is markedly stimulated by inhibitors of protein and RNA synthesis, including cycloheximide, puromycin and actinomycin D. Our original interpretation of this finding was that inhibiting protein synthesis led to metabolic changes in the cell which converged upon the intracellular pathway used by laminin to stimulate neurite formation. Alternatively, Preston et al. (1987, Proc. natn. Acad. Sci. U.S.A. 84, 5247–5251) suggested that cycloheximide acts by inhibiting neurite retraction. In order to resolve these conflicting interpretations, we decided to re‐examine the effects of cycloheximide using time lapse analyses of NG108‐15 cells grown on laminin, together with morphometric analyses of cultures fixed at 1.5 or 3 hr after plating. Cycloheximide was found to cause both an increase in neurite initiation within the first 1.5 hr after plating, and a decrease in the occurrence of neurite retractions between 1 and 3 hr. Investigators should be aware of the effects of biosynthetic inhibitors on neurite growth, since these agents are employed widely in studies of cultured neural cells, particularly those involving receptor metabolism and phenotypic differentiation.
Perinatal morphine exposure alters peptidergic development in the striatum
Authors: Tenconi Beatrice , Donadoni Maria Luisa , Finco Cristina , Germani Elena , Malosio Maria Luisa , Di Giulio Anna Maria , Mantegazza Paolo , Gorio Alfredo
Published: April 2003

It has been reported that perinatal exposure to opiates affects mRNA synthesis, body growth and brain development in mammals, including humans. We have observed that morphine administration in drinking water during the perinatal period alters peptide development in the striatum of the rat. There is a marked increase in substance P and met‐enkephalin content, the latter is maintained even at 30 days postnatally. The transient increase or earlier maturation of substance P content is correlated by a more precocious axon terminal organization as revealed by immunocytochemical staining. The increased metenkephalin content is correlated by a higher abundance of preproenkephalin A mRNA and this correlation is particularly evident at 15 days postnatally. At earlier times both northern blotting and in situ hybridization techniques fail to show any significant difference between control and morphine exposed rats, likely because the peptide content is not very different in the two groups or at least the gap is not as wide as at later times.
Aging‐related changes in IGF‐II and c‐fos gene expression in the rat brain
Authors: Kitraki Efthimia , Bozas Evangelos , Philippdis Helen , Stylianopoulou Fotini
Published: April 2003

The protein products of growth factor genes such as IGF‐II and cellular oncogenes such as c‐fos are believed to be necessary for the support of normal neuronal function. Steady‐state levels of c‐fos and IGF‐II mRNA were determined in the brain of young and old rats, using Northern analysis. Both RNAs were found to be decreased in the brain of aged rats. Age‐related decrease was detected in the hippocampus, hypothalamus, striatum, cerebral cortex and cerebellum, for IGF‐II mRNA, and in the cerebral cortex and cerebellum for c‐fos mRNA. Furthermore, changes in the degree and pattern of DNA methylation were noted at both gene loci, in the aged rat brain. Our results could reflect changes at the genomic level possibly related to the process of aging and the accompanying decline in brain function.
Voltage sensitive calcium channels mark a critical period in mouse neurodevelopment
Authors: Litzinger M.J. , Grover B.B. , Saderup S. , Abbott J.R.
Published: April 2003

Voltage sensitive calcium channel (VSCC) probes 125I‐ω‐GVIA Conotoxin (ω‐GVIA), (+)‐[5‐methyl‐3H]‐PN200‐110 (3H‐PN200), and 3H‐Nimodipine were bound to developing Swiss Webster mouse whole brain from postnatal days 3 to 24. 125I‐ω‐GVIA binding, thought to be presynaptic, showed a 50% increase between days 11 and 14. 3H‐dihydropyridine binding, thought to be postsynaptic, showed spike patterns when measured developmentally. 3H‐PN200 binding showed a >150% increase between days 11 and 15. 3H‐Nimodipine binding showed a >100% increase between days 11 and 14. Depolarization‐induced 45Ca fluxes also increased between days 8 and 16 by >500%. The dramatic increases indicated by these binding data correspond to a critical period described by Himwich (Int. Rev. Neurobiol. 4, 117, 1962) between postnatal days 11 and 14 in Swiss Webster mice; during this critical period, dendrites exhibit rapid outgrowth, sensory modalities come on line, EEG patterns mature, and the cortex reaches adult proportions. We conclude from these data that the increase in VSCC activity parallels a critical period in the development of the central nervous system in Swiss Webster mice.
Migration of purified embryonic motoneurons grafted into adult mouse CNS
Authors: Ruiz‐Flandes P. , Demierre B. , Mattenberger L. , Kato A.C.
Published: April 2003

Embryonic motoneurons were fluorescently‐labelled with carbocyanine (diI) by means of retrograde transport and then grafted into the adult mouse spinal cord (L2) and brain (striatum) for 2–10 weeks. The motoneurons were grafted either following purification on the fluorescence‐activated cell sorter or in the presence of embryonic glial cells and interneurons from the spinal cord. In both conditions of grafting, motoneurons were found to survive and develop in both grey and white matter and were found to migrate long distances in both regions of the central nervous system.

Migration of neurons after grafting remains a controversial issue, therefore we have discussed the work of other groups that have described the same phenomenon.
Co‐expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study
Authors: Janeczko Krzysztof
Published: April 2003

Changes in the distribution of proliferating astrocytes expressing glial fibrillary acidic protein (GFAP) and/or vimentin were examined in the injured cerebral hemisphere in adult mice. The injury was followed by [3H]thymidine injections at different time intervals. The brain sections were doubly immunostained for GFAP and vimentin and subjected to autoradiography. In that way three cell types were distinguished immunocytochemically: (1) astrocytes co‐expressing glial fibrillary acidic protein (GFAP) and vimentin (2) astrocytes expressing only GFAP (3) astrocyte‐like cells expressing vimentin. Thereafter, numbers of immunopositive and autoradiographically labelled cells and their locations within the region of injury were recorded at each stage of the experiment. Two hours as well as 1 day after the injury proliferation of GFAP‐positive astrocytes and of those co‐expressing GFAP and vimentin could only be seen as statistically insignificant phenomena. On day 2 the reactive proliferation of each immunocytochemically defined cell type was already maximal, then gradually decreased and its last signs were recorded on day 8. On day 2, among all the proliferating GFAP‐positive astrocytes, 67.2% were also vimentin‐positive. Later, the proportion declined to 50.7% and 38.5% on days 4 and 8, respectively. The labelled astrocyte‐like vimentin‐positive cells were located closest to the lesion margins. In comparison, the astrocytes co‐expressing GFAP and vimentin and those expressing exclusively GFAP, occupied regions progressively farther from the lesion site. At the initial stages of the response to injury, vimentin expression in cells starting their reactive proliferation did not precede the expression of GFAP. This was considered as an argument against a hypothesis that reactive astrocyte division induces a two‐stage increase in the cytoskeletal protein level in which synthesis of vimentin precedes that of GFAP.
CSF‐1 expression is upregulated in astrocyte cultures by IL‐1 and TNF and affects microglial proliferation and morphology in organotypic cultures
Authors: Shafit‐Zagardo Bridget , Sharma Nishi , Herman Joan W. , Bornstein Murray B. , Brosnan Celia F.
Published: April 2003

Astrocytes produce factors that control the growth and differentiation of many cell types within the CNS as well as play a role in the generation of the immune response. The extent to which these two functions interact has received less attention. We now report that astrocyte cultures established from rat brain endogenously express mRNA and low levels of secreted biologically active protein for the monocyte growth and differentiation factor colony stimulating factor‐1 (CSF‐1). Exposure of astrocytes to interleukin‐1 (IL‐1) and/or tumor necrosis factor (TNF) upregulated the expression of CSF‐1 mRNA and protein. Following treatment with 100 U/ml of TNF, IL‐1, or TNF+IL‐1, maximum CSF‐1 mRNA expression was observed at 3 hr. In the presence of IL‐1 an increase in biologically active CSF‐1 was detected in the astrocyte conditioned medium at 6 hr. These data indicate that the expression of CSF‐1 by astrocytes can be modulated by exposure to the cytokines IL‐1 and TNF. To determine whether CSF‐1 provides a mitogenic signal for microglia during development, mouse spinal cord organotypic cultures were exposed to recombinant mouse CSF‐1 (rmCSF‐1), resulting in proliferation of microglia by 7 days and an increase in the number of ramified microglia over ameboid microglia by 14 days.
Effects of hyperactivity of the maternal hypothalamic‐pituitary‐adrenal (HPA) axis during pregnancy on the development of the HPA axis and brain monoamines of the offspring
Authors: Fameli M. , Kitraki E. , Stylianopoulou F.
Published: April 2003

Offspring of mothers with adrenal hyperactivity during pregnancy have been reported to have changes in brain monoamines and altered emotional, reactive, sexual and maternal behavior. Since the hypothalamic‐pituitary‐adrenal (HPA) axis is known to be involved in the expression of such behaviors and is itself under monoaminergic control, we examined the development of the HPA axis and brain monoamines in pups whose mothers had adrenal hyperactivity, reflecting administration of ACTH during the last third of their pregnancy. The adrenals of the experimental animals weighed less and had aberrant morphology. The abnormal histology was more pronounced in the adrenals of the experimental females than of the males, suggesting that females were more vulnerable to the prenatal treatment. In both experimental males and females, basal plasma corticosterone levels were higher compared to the controls, while after exposure to stress, experimental animals attained lower plasma corticosterone levels than the controls. In the brain of the experimental animals, dopaminergic activity appeared to be decreased, while serotonergic activity increased. Our results indicate that the prenatal treatment affected brain development in the offspring and as a consequence programmed the developing HPA axis in such a way as to hyperfunction under basal conditions, leading to its exhaustion and its inability to react properly to stress.
Expression of neurotrophin genes in human fibroblasts: Differential regulation of the brain‐derived neurotrophic factor gene
Authors: Cartwright M. , Mikheev A.M. , Heinrich G.
Published: April 2003

Brain‐derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin‐3 (NT‐3) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. We previously reported that BDNF and NGF gene expression are differentially regulated in mouse L929 fibroblasts. Here we examine expression of these three neurotrophins in human fibroblasts. Northern blots detected BDNF and NT‐3 mRNAs in fibroblasts derived from lung (WI‐38), calvarium and foreskin. WI‐38 cells and foreskin fibroblasts expressed 1.6 kb as well as 4 kb BDNF mRNAs whereas only the smaller BDNF mRNA was detected in calvarium fibroblasts. NGF mRNA was present in foreskin and calvarium but not lung fibroblasts. In WI‐38 cells serum treatment increased levels of BDNF mRNA within 2 hr. Cycloheximide did not inhibit the increase. Treatment with 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) transiently suppressed BDNF mRNA. Treatment with both serum and TPA first stimulated and then transiently suppressed BDNF mRNA. TPA and/or serum did not significantly affect BDNF mRNA in calvarium fibroblasts. These results show that human fibroblasts derived from different tissues express and regulate neurotrophin genes differentially.
Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina
Authors: Carri Nestor G. , Richardson Peter , Ebendal Ted
Published: April 2003

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E 18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose‐dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.
A reverse transcription‐polymerase chain reaction study of p75 nerve growth factor receptor gene expression in developing rat cerebellum
Authors: Chu‐Kuang Chen , Kinsman Stephen L. , Holtzman David M. , Mobley William C. , Johnston Michael V.
Published: April 2003

The actions of the neurotrophins are mediated through specific receptors. Nerve growth factor (NGF), the prototypic neurotrophin, binds to receptors of both high and low affinity. A protein 75 kDa in size (p75NGFR) binds NGF, as well as brain‐derived neurotrophic factor and neurotrophin 3, with low affinity. Recent investigations suggest that this protein may also be a component of the high affinity NGF receptor complex. To study gene expression of the p75NGFR molecule, we used a sensitive reverse transcription‐polymerase chain reaction (RT‐PCR) assay to measure levels of its messenger RNA (mRNA) in small samples of total RNA. The assay is based on using a shortened p75NGFR cRNA as an internal RNA standard to control for variability in reverse transcription and polymerase chain amplification. We measured p75NGFR mRNA levels in the rat cerebellum during ontogeny to further study the transient developmental increase in receptor gene expression known to occur in this brain region during the early postnatal period. We found that p75NGFRmRNA levels were most abundant at postnatal day 2, and then declined to lower levels throughout postnatal development and in the adult. Northern blot analysis of the same total RNA samples used in our RT‐PCR assay verified that p75NGFR expression is highest in the early postnatal period. These results confirm those of previous studies accomplished with much larger amounts of RNA using ribonuclease protection or northern blot assays. The use of an RT‐PCR assay that utilized an internal standard also controls against changes in RNA complexity which can affect the measurement of message abundance across developmental stages. These results again suggest that p75NGFR may play an important role in the postnatal development of cerebellum in rats
Olfactory ensheathing glia and platelet‐derived growth factor B‐chain reactivity in the transplanted rat olfactory bulb
Authors: Kott J.N. , Westrum L.E. , Raines E.W. , Sasahara M. , Ross R.
Published: April 2003

Using a monoclonal antibody against the B‐chain of platelet‐derived growth factor as a marker, we have examined the behavior of olfactory ensheathing glia in the normal and transplanted rat olfactory bulb. In the normal postnatal olfactory bulb, these glia are found to ensheath the bundles of incoming primary olfactory nerve fibers as well as those in the olfactory nerve layer. Olfactory marker protein antibody was used to identify the olfactory nerve proper. Within the transplant, the same glia: (1) ensheath bundles of both primary olfactory and non‐primary olfactory axons, (2) ensheath axonal bundles deep within the donor tissue, and (3) eventually permit radiation of individual axons from bundles to surrounding neuropil.

We believe that ensheathing glia (being rich in growth‐related factors and extracellular matrix molecules) may be useful in providing trophic support and guidance for the reconstruction of developmentally or traumatically damaged neuronal pathways not directly related to the olfactory system. The evidence presented here indicates that ensheathing glia are capable of existing in deep brain areas and ensheathing other than primary olfactory axons. The special molecular characteristics of these glia along with the morphological findings presented here provide a foundation for further studies of these unique glia and their potential utility in the restoration of damaged neural pathways.