bluuebunny/crossref_metadata_2025 huggingface dataset:
Taurine effect on neuritic growth from goldfish retinal explants
Authors: Lima L. , Matus P. , Drujan B.
Published: April 2003

Explants from retinas from which the optic nerve was previously crushed exhibited neuritic outgrowth after 4–5 days in culture, and up to 10 days in the presence of the same nutrient medium. Intact retinas did not exhibit vigorous outgrowth. Taurine added in the medium to post‐crush explants accelerated the emission of neurites in 1–2 days, and increased the nerve growth index in a concentration‐dependent manner up to 4 mM. Greater concentration did not produce any effect on the nerve growth index. The effect of taurine was produced between 0 and 5 days in culture; from day 5 to day 10 explants did not exhibit further growth in the presence of the amino acid. However, post‐crush control explants exhibited growth between 5 and 10 days. Taurine effect is blocked in the absence of fetal calf serum, and is reduced in the presence of dialysed serum. Providing new medium on days 1 and 2, but not later, elevated the nerve growth index with respect to expiants regenerating in the same medium since day 0. In addition, taurine supplied at various days after plating produced the same pattern of growth as that observed by adding fresh medium, but the nerve growth index was statistically significantly greater in the presence of the amino acid. The mechanisms for these observations remain to be elucidated.
The effects of intrauterine growth retardation on the development of the Purkinje cell dendritic tree in the cerebellar cortex of fetal sheep: A note on the ontogeny of the Purkinje cell
Authors: Rees Sandra , Harding Richard
Published: April 2003

The development of the fetal sheep cerebellum at 80, 100, 120 and 140 days gestation (term = 146 days) and 3 months postnatally was studied using Nissl stained sections and rapid Golgi preparations. The most rapid expansion of the Purkinje cell dendritic tree occurred between 100 and 120 days of gestation (5–6 fold increase in area). By 140 days it had acquired its adult form after which time growth continued mainly in the vertical direction. The effects of intrauterine growth retardation on the growth of granule and Purkinje cell dendrites in the cerebellar cortex of fetal sheep (140 days) were investigated in Golgi preparations. Compared with control cerebella the length (but not the number) of granule cell dendrites was reduced by 14% (P<0.01); the area of the Purkinje cell dendritic field was reduced by 20% (P<0.01); the branching density was reduced by 8% (P<0.01); the total branch length was reduced by 27% (P<0.002); the density of dendritic spines per row was not affected. These factors resulted in a decrease of 26% (P<0.002) in the total number of dendritic spines per row per Purkinje cell.

These findings show that the growth of granule cell dendrites and the Purkinje cell dendritic tree have been significantly affected by chronic intrauterine deprivation. Such structural abnormalities could affect the pattern of neuronal connectivity and could be associated with functional deficits.
Developmental expression of proenkephalin a mRNA and phenylethanolamine n‐methyltransferase mRNA in foetal sheep adrenal medulla
Authors: Wan David C.‐C. , Choi Chung‐lit , Livett Bruce G.
Published: April 2003

The ontogenic expression of proenkephalin A (ProEnk A) mRNA and phenylethanolamine N‐methyltransferase (PNMT) mRNA was examined in the foetal sheep adrenal medulla by the use of specific oligodeoxyribonucleotide probes. Northern blot analysis of RNA extracts from foetal adrenals demonstrated that ProEnk A mRNA was expressed as early as 60 days of gestation, a time at which the foetal adrenal is not functionally innervated. In situ hydridization on sections of foetal adrenals revealed that at 110–140 days gestation ProEnk A mRNA was expressed in chromaffin cells at the outer margin of the adrenal medulla but at earlier stages of gestation (e.g. 95 days) appeared to be expressed homogeneously throughout the whole of the adrenal medulla. In comparison, PNMT mRNA was expressed preferentially in cells at the outer margin of the adrenal medulla from the earliest stage detectable. Both PNMT mRNA and ProEnk A mRNA co‐localized in cells at the outer margin of foetal adrenal of late gestations (110–140 days), a similar pattern to that seen in the adult adrenal medulla. These results indicate that, as with adult animals, in foetuses of late gestation, adrenal enkephalins are co‐stored within adrenaline cells. It is likely therefore that enkephalins are co‐released from the foetal adrenal with adrenaline in response to intra‐uterine stress.
Synaptic density of caudate‐putamen and visual cortex following exposure to ethanol in utero
Authors: Lancaster F. , Delaney C. , Samorajski T.
Published: April 2003

Pregnant Long‐Evans rats were fed a liquid diet containing ethanol (30% of total calories) during days 3–19 of gestation. Controls were given ad libitum access to liquid diet lacking ethanol, or pair‐fed isocaloric amounts based on consumption by the animals in the ethanol group. Brain development of female offspring was evaluated by analysis of electron micrographs of caudate‐putamen and visual cortex. Numbers of presynaptic terminals and synaptic junctions (synaptic density) per unit area were compared for 14‐ and 28‐day‐old offspring of dams from the three treatment groups. Synaptic density of the caudate‐putamen and visual cortex was not affected by ethanol at 14 or 28 days. Although exposure to ethanol during a period comparable to the first two trimesters of human development with minimal or no undernutrition did not affect numerical density of synapses in visual cortex or caudateputamen, synaptogenesis of caudate‐putamen was altered in offspring of pair‐fed animals.
Sexual differentiation of mesencephalic neurons in vitro: Effects of sex and gonadal hormones
Authors: Engele Jürgen , Pilgrim Christoph , Reisert Ingrid
Published: April 2003

In order to study the influence of gender on the development of transmitter uptake by dopaminergic neurons, dissociated cell cultures were raised separately from male and female midbrains of gestational day 14 rats. It was ascertained by use of specific inhibitors and by autoradiography that the uptake of [3H]dopamine was restricted to neurons immunoreactive for tyrosine hydroxylase and that these neurons have dopaminergic properties. The uptake capacity was higher in male than in female dopaminergic neurons by a factor of 1.5. This sexual dimorphism in dopamine uptake was present in cultures of tissue removed before the perinatal rise of testosterone occurs in males and was present even in the absence of hormonal additives to the culture medium. It therefore appears to be independent of the presence of gonadal steroids. It occurred likewise in cultures raised with serum‐supplemented and serum‐free medium, which may indicate that glia are not decisive in generating these differences. In addition, sexual differences were found regarding hormone responsiveness. Whereas testosterone and dihydrotestosterone were ineffective, estradiol was seen to promote dopamine uptake in female but not in male neurons. The results would suggest that mesostriatal and/or mesolimbic dopaminergic systems assume an early role in the development of some sexual dimorphisms of the brain.
Premetamorphic effects of thyroid hormones on tadpole sensory ganglia
Authors: Ando Masato , Hammerschlag Richard
Published: April 2003

Tadpoles at premetamorphic stages of development were used to compare the precocious responses of lumbar dorsal root ganglia (DRG) and hindlimb bud (tissues destined for growth) with the responses of tail DRG and tail muscle (tissues destined for resorption) following exogenous administration of triiodothyronine (T3) and thyroxine (T4). Responses to intraperitoneal (i.p.) hormone treatment were assessed at varying times by injection of [3H]leucine i.p. and determination of 3H‐labeled protein in tissues after an additional 1.5 hr. Incorporation of [3H]leucine in lumbar DRG and hindlimb bud was markedly stimulated by either hormone. T3 and T4 effects were both maximal at 0.3 nmol/g body wt although, as examined in lumbar DRG, the response to T4 was more rapid and of lesser magnitude than that to T3. By contrast, incorporation in tail DRG and tail muscle was significantly depressed in response to T3 and was unaffected by T4. Co‐injection of T3 and T4 (either 1:1 or 1:6 as occurs during the peak surge of circulating thyroid hormones during metamorphic climax) did not produce an additive effect; the hindlimb bud response was reduced while the lumbar DRG, tail DRG and tail muscle responses to the individual hormones were virtually eliminated. The present data suggest that the responses of lumbar and tail sensory neurons to thyroid hormones parallel the responses of their peripheral target tissues.
Developmental changes in the chemosensitivity of rat brain synaptoneurosomes to excitatory amino acids, estimated by inositol phosphate formation
Authors: Guiramand Janique , Sassetti Isabelle , Recasens Max
Published: April 2003

The evolution of excitatory amino acids‐(EAA) stimulated inositol phosphates (IPs) turnover during postnatal development was investigated in synaptoneurosomes prepared from rat forebrains. The two main EAA agonists which induce the IPs synthesis were quisqualate (QA) and N‐methyl‐d‐aspartate (NMDA). The QA and NMDA stimulations of IPs formation present a particular developmental pattern, characterized by an active phase during rat synaptogenesis. The QA‐evoked IPs accumulation peaked in synaptoneurosomes prepared from 8‐day‐old rat forebrains while that evoked by NMDA peaked in synaptoneurosomes from 12‐day‐old rats. These two developmental patterns are specific of the EAA agonists since the other various neuroactive substances tested (carbachol (Carb), noradrenaline, and high concentrations of potassium) induced an IPs accumulation, which increases during development and reaches a maximum in synaptoneurosomes of adult animals. Aging leads to a decrease in the capability of EAAs and muscarinic agonists to stimulate IPs formation in synaptoneurosomes, whereas the stimulation of IPs turnover by noradrenaline remains constant.

Taken together, these results suggest that EAAs play a key role during brain development by sequentially activating two receptor subtypes, a new QA receptor, and a NMDA receptor, linked to the phosphoinositide metabolism. They may also indicate that these EAA‐induced IPs responses are related to neuronal plastic events, the amplitude of which decreases with aging.
The effects of intrauterine growth retardation on the structural development of cranial nerves (optic, trochlear) in fetal sheep
Authors: Rees Sandra , Clark Malcolm , Snowden Michael , Harding Richard
Published: April 2003

A quantitative morphometric study of the development of myelinated fibres in the optic and trochlear nerves has been made in growth‐retarded fetal sheep at 140 days gestation (term = 146 days). Intrauterine growth retardation was induced as a result of the reduction of placental mass, by prior removal of placentation sites in six ewes. In the optic nerve (central nervous system) the mean diameter of myelinated fibres was not significantly reduced but the thickness of the myelin sheath relative to axon diameter was disproportionately reduced. In the trochlear nerve (peripheral nervous system) there was a significant reduction of 23% (p < 0.01) in the mean diameter of myelinated fibres; however the normal axon:myelin ratio was maintained. The total number of myelinated fibres in the trochlear nerve did not differ between the normal and growth‐retarded group, indicating that there was not a greater than normal incidence of cell death during intrauterine growth retardation in the nucleus of the trochlear nerve.

The differential effect of intrauterine growth retardation on myelination in the central and peripheral nervous systems suggests that chronic intrauterine deprivation affects oligodendrocyte activity but does not markedly affect the capacity of Schwann cells to produce myelin.
Localization of dipeptidylpeptidase IV and alkaline phosphatase in developing spinal cord meninges and peripheral nerve coverings of the rat
Authors: Haninec Pavel , Grim Miloš
Published: April 2003

Localization of dipeptidylpeptidase IV was studied in the spinal cord meninges and peripheral nerve coverings of fetal and postnatal rats. In the same sections, the localization of alkaline phosphatase was monitored.

In the prenatal period, dipeptidylpeptidase (DPP) IV activity in the differentiating meninges appeared at the time of cerebrospinal fluid spaces formation (on day 16 in the cervical region and on day 18 in the lumbar region).

In adult animals DPP IV was found in cells of those meningeal lamellae which delineated the cerebrospinal fluid spaces (the outer, intermediate and inner lamellae), in the perineurium, in Schwann cells and in some fibroblasts of the bulk of dura mater. It is suggested that DPP IV plays a role in the metabolism of neuropeptides by their interaction with cerebrospinal fluid.

Alkaline phosphatase activity was detectable earlier than DPP IV activity. Positivity was first observed in some cells of the meninx primitiva and, later on, in the ectomeninx and also in the differentiating endomeninx where it disappeared postnatally.

The developing ectomeninx exhibited activities of both enzymes. Alkaline phosphatase occupied its external layers, while DPP IV was localized in its inner layers. This enzymatic heterogeneity of the ectomeningeal layers suggests that the ectomeninx gives rise not only to dura mater (which in adult animals exhibits alkaline phosphatase activity) but also to the outer arachnoid layer (positive for DPP IV in adult rats).
Cortical neuron sensitivity to neurotransmitters following neonatal noradrenaline depletion
Authors: Mirmiran M. , Dijcks F. A. , Bos N. P. A. , Gorter J. A. , Van der Werf D.
Published: April 2003

In order to test the functional significance of rapid eye movement (REM)‐sleep and noradrenergic activity for cerebral cortex maturation, rat pups were daily injected with clonidine from 8 to 21 days of life. Previous studies have shown that this treatment reduces the amount of time spent in REM‐sleep and the level of noradrenaline turnover in the brain. For long‐term consequences of such treatment in adulthood, cortical neuron responses to micro‐iontophoretically applied neurotransmitters were studied. No significant differences were found in the single cell responses to glutamate, GABA or noradrenaline in the cerebral cortex of clonidine treated rats as compared with age matched controls. However, the magnitude of GABAergic depression of glutamate induced neuronal responses was greater in the clonidine than in the control group.
Structural and neurochemical effects in mouse cerebellum following neonatal methylazoxymethanol and 6‐hydroxydopamine treatment
Authors: Luthman J. , Eriksdotter‐Nilsson M. , Jonsson G.
Published: April 2003

The effects of neonatal treatment with the antimitotic agent methylazoxymethanol and the catecholamme neurotoxin 6‐hydroxydopamine on cerebellar morphology and monoamine innervation in the N.M.R.I. mouse has been studied. Methylazoxymethanol (25 mg/kg s.c.) treatment induced a cerebellar weight reduction of 40% as observed in the adult stage, while other CNS regions analysed were unaffected. An obvious atrophy of the cerebellar cortex was found, with an irregular distribution of the Purkinje cells, while Bergmann glia fibers deviated from their normal radial configuration and showed a tendency to form clusters. A 65% increase of tyrosine hydroxylase immunoreactive fiber density was found in the cerebellar cortex and 3H‐5‐hydroxytryptamine in vitro synaptosomal uptake was increased by 55%. Noradrenaline and 5‐hydroxytryptamine concentrations in the cerebellum increased by 50 and 30%, respectively, whereas the total content of both neurotransmitters in cerebellum was approximately unchanged after methylazoxymethanol treatment. A significant reduction in total cerebellar in vitro binding of 3H‐WB‐4101 and 3H‐dihydroalprenolol was also found, indicating compensatory receptor alterations following methylazoxymethanol treatment. The effect of combined treatment of methylazoxymethanol and the neurotoxin 6‐hydroxydopamine (50 mg/kg s.c., day 1) showed a very pronounced reduction of noradrenaline concentration in cortex cerebri, while the noradrenaline concentration in cerebellum was increased by 185% and the tyrosine hydroxylase immunoreactive fiber density by 125%, indicating an additional relative hyperinnervation of cerebellar noradrenaline fiber due to a “pruning effect” of the 6‐hydroxydopamine treatment. The results imply a relatively rigid development of terminal arborization of central nervous system monoamine neurons, relatively independent of neuronal and glial arrangement in the target area.
A developmental study of soluble L1
Authors: Nybroe Ole , Dalseg Anne‐Marie , Bock Elisabeth
Published: April 2003

L1 is a neural cell adhesion molecule expressed by neurons and it is involved in cell interactions during axon elongation and fasciculation. L1 from rat brain consists of a membrane‐inserted Mr 200,000 polypeptide from which two polypeptides of Mr 180,000 and Mr 140,000 can be derived. These latter polypeptides appear both as membrane‐associated and as soluble molecules. In this report, both total and soluble L1 in rat brain have been quantified by an enzyme‐linked immunosorbent assay. The amount of total L1 per gram brain varies with postnatal age showing a peak value at postnatal day 7. The variation in soluble L1 coincides with the changes in total L1. Thus, soluble L1 constitutes ca 2% of total L1 at all ages investigated. The soluble Mr 140,000 and 180,000 L1 polypeptides are also present in cerebrospinal fluid. Studies of membrane L1 catabolism in cultured fetal rat brain neurons show that the half‐life of membrane L1 is less than 24 hr. As a part of membrane L1 catabolism, small amounts of soluble L1 polypeptides are released to include cell surroundings.
Spontaneous neuronal firing patterns in the occipital cortex of developing rats
Authors: Corner M. A. , Mirmiran M.
Published: April 2003

Spontaneous action potentials have been recorded in the cerebral cortex of developing rats under chloral hydrate anaesthesia. These “spike‐trains” were subjected to a multivariate analysis of parameters describing different aspects of temporal patterning within each train. Three parameters which reflected clustering of action potentials in, respectively, the range of: (i) milliseconds (the modal interval, derived from the interval histograms); (ii) seconds (the mean “burst period”, i.e. the time elapsing between the onset of successive clusters of relatively short intervals); and (iii) minutes (the coefficient of variation for the number of spikes in consecutive 2 min time bins) showed distinctive developmental patterns.
Early appearance of tyrosine hydroxylase immunoreactive cells in the mesencephalon of mouse embryos
Authors: Di Porzio Umberto , Zuddas Alessandro , Cosenza‐Murphy Dano B. , Barker Jeffery L.
Published: April 2003

The development of mesencephalic catecholaminergic neurons in the embryonic and fetal mouse was analysed in tissues fixed with 5% acrolein using polyclonal rabbit antibodies against tyrosine hydroxylase (TH), the first enzyme in catecholamine synthesis. The first TH positive cells were identified as early as day 8.5–9 of gestation and some expressed TH while apparently still migrating from the proliferative layer. The number of catecholamine cells increased dramatically by embryonic day 9.5–10; at gestation days 10.5–11 numerous TH positive cells bearing many neurites were localized in the ventral part of the mesencephalon but they were not yet separated into two different groups (A9 and A10). After 13 days of gestation two separate catecholaminergic groups could be visualized, although many TH positive cells with long neurites (putative dopaminergic neurons) could still be seen at the edges of the ventricle, and appeared to be moving towards the ventral mesencephalon.

On the basis of these results the possibility that catecholamine cells that are produced early during the development of the midbrain may have neurotrophic and/or morphogenetic roles is discussed.
Immunocytochemical detection of glutamate decarboxylase in the postnatal developing rat organ of corti
Authors: Merchan‐Perez Angel , Gil‐Loyzaga Pablo , Eybalin Michel
Published: April 2003

Using a fluorescent avidin‐biotin technique, we have immunolocalized the GABA synthesizing enzyme, glutamate decarboxylase, in postnatal developing and adult rat organs of Corti. At birth, the glutamate decarboxylase‐like immunoreactivity is already present in the basal turn below the inner hair cells, i.e. within lateral olivocochlear efferent fibers of the inner spiral bundle. In the apical turn, the inner spiral bundle displays an immunoreactivity as early as postnatal day 3. Only the outer hair cells of the upper second turn and apex receive fibers immunostained for glutamate decarboxylase that most probably belong to the medial olivocochlear efferent innervation. They first appear at this level at postnatal day 15. Within these two regions of the organ of Corti, the glutamate decarboxylase‐like immunoreactivity reaches an adult‐like pattern at postnatal days 17–18. These results strengthen the hypothesis that GABA is a putative neurotransmitter that could be used by subpopulations of the two olivocochlear innervations. They also suggest that GABA either plays a neurotrophic function or participates in the regulation of the first cochlear potentials at the level of lateral efferent synapses.
Sprouting by isolated Helisoma neurons: Enhancement by glutamate
Authors: Bulloch A. G. M. , Hauser G. C.
Published: April 2003

We tested glutamate for its ability to modulate neurite outgrowth from isolated neurons of the adult snail, Helisoma trivolvis. Although glutamate did not induce neunte outgrowth from neurons maintained in defined medium, nevertheless it showed a dose‐dependent ability to enhance the activity of conditioned medium. We concluded that glutamate can enhance the release and/or activity of CNS derived sprouting factor(s) present in conditioned medium.

The general conclusion to be drawn from this study is that the ability of a neurotrophic factor(s) to promote neunte outgrowth can be regulated by a neurotransmitter. This mechanism may be important in the regulation of trophic factors in the adult nervous system.
GABA agonist induced changes in ultrastructure and GABA receptor expression in cerebellar granule cells is linked to hyperpolarization of the neurons
Authors: Belhage Bo , Hansen Gert H. , Schousboe Arne
Published: April 2003

GABA has been shown to exert a neurotrophic like activity by enhancing the morphological and functional maturation of neurons. Mechanisms involved in this effect of GABA are largely unknown but since GABA has been shown to mediate a hyperpolarizing action on neurons it can be assumed that this action might be important. In order to investigate this possibility, the ability to mimic the trophic actions of GABA of different agents known to influence the membrane potential or the GABA gated chloride channels was studied. Hence, GABA receptor expression as well as the ultrastructure of cerebellar granule cells were monitored after exposure of the cells in culture to either bromide, valinomycin or picrotoxin. It was found that cells which at early developmental stages (4 days in culture) were exposed to bromide or valinomycin expressed low affinity GABA receptors similar to cells treated with the GABA agonist 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP). This was in contrast to untreated cultures or cultures treated with both THIP and picrotoxin which expressed only high affinity GABA receptors and no low affinity receptors. In cultures at more mature stages (14 days in culture) bromide treatment did not lead to formation of low affinity GABA receptors. Studies of the ultrastructure of the cells (4‐day‐old cultures) showed that exposure to bromide or valinomycin mimicked the ability of THIP to enhance the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus, vesicles and coated vesicles. Again, in 14‐day‐old cultures treatment with bromide had no effect on the ultrastructure. Exposure of the cells to picrotoxin significantly reduced the ability of THIP to induce the increase in the density of the organelles mentioned above. Altogether the results strongly suggest that the hyper‐polarizing action of GABA via the chloride channels coupled to high affinity GABA receptors is the permissive signal for initiation of further reactions involved in the trophic and differentiating actions of GABA.
Antioxidant effect of retinoic acid on PC12 rat pheochromocytoma
Authors: Jackson George R. , Morgan Brent C. , Werrbach‐Perez Karin , Perez‐Polo J. Regino
Published: April 2003

Retinoic acid is a naturally occurring metabolite of vitamin A that influences the differentiation of a variety of neural cells in vitro. In the LA‐N‐1 human neuroblastoma line, retinoic acid treatment increases the binding of nerve growth factor (Bmax). The purpose of this study was to examine the effects of retinoic acid on PC12 rat pheochromocytoma, a neural crest‐derived cell line that can be induced to express a sympathetic neuroblast‐like phenotype by nerve growth factor treatment. In contrast to the differentiating effects of nerve growth factor, retinoic acid treatment of PC12 cells had a negligible effect on cellular morphology. However, treatment with retinoic acid enhanced the survival of PC12 cells following oxidative injury generated by H2O2 treatment in a manner that is qualitatively similar to that observed after nerve growth factor treatment. Also, there was an increase in 125I‐nerve growth factor binding activity in solubilized PC12 membrane preparations derived from retinoic acid‐treated PC12 cells. These data suggest that retinoic acid may play a role in neuronal development and in neuronal injury by stimulating the ability of neurons to cope with oxidative stress and/or by enhancing neuronal responsiveness to trophic factors such as the nerve growth factor.
Development of binding sites for excitatory amino acids in cultured cerebral cortex neurons
Authors: Wahl P. , Honoré T. , Drejer J. , Schousboe A.
Published: April 2003

Binding of [3H]glutamate, [3H]AMPA (RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolo‐propionate) and [3H]kainate was investigated in membranes prepared from cerebral cortex of 4‐day‐old and adult mice and from cerebral cortex neurons cultured for different periods of time (2, 4, 8 and 14 days). For all ligands, the number of binding sites increased as a function of development both in vivo and in culture. A significant number of binding sites for the ligands could be demonstrated on the cultured neurons already after 2 days in culture. Scatchard analysis of the binding data showed a single population of binding sites for glutamate (KD≈200 nM) and kainate (≈6 nM) regardless of the developmental stage in vivo or in culture. In case of [3H] AMPA binding two binding sites with KD values of ≈6 nM and 100–200 nM could be demonstrated both in vivo and in culture. Binding of [3H]glutamate to cultured neurons could be displaced by N‐methyl‐d‐aspartate (100 μM) and quisqualate (3 μM) in an additive manner but d,l‐4‐aminophosphonobutyrate (100 μM) had no effect. AMPA binding to cultured neurons was much more (40‐fold) sensitive than kainate binding to the newly developed AMPA selective antagonist NBQX (2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo(F)quinoxaline) indicating that kainate and AMPA bind to independent binding sites. Monitoring membrane potentials in the cultured neurons using the lipophilic cation TPP+ (tetraphenylphosphonium) it was demonstrated that potassium (55 mM) as well as glutamate, AMPA and kainate (100 μM) could depolarize the neurons both at early (2 days) and late (9 days) developmental stages in culture. The demonstration of functionally active receptors for the 3 excitatory amino acids in both immature (2 days in culture) and mature (8–9 days in culture) neurons is discussed in the light of previous studies of the development as a function of the culture period of effects of excitatory amino acids in neurons. It is concluded that no simple correlation exists between expression of binding sites for the excitatory amino acids and their ability to induce cytotoxicity and neurotransmitter release.
Effects of prostaglandin E2 and cyclooxygenase inhibitors on clustering and level of nicotinic acetylcholine receptor in mouse myotubes co‐cultured with spinal cord explant
Authors: Kimura I. , Nakagawa M. , Kobayashi S. , Kimura M.
Published: April 2003

The clustering and level of nicotinic acetylcholine receptor (n‐AChR) in cultured mouse myotubes are negatively controlled by endogenous phospholipase A2 (PLA2) (Kimura et al., Int. J. Devl. Neurosci.5, 127–133, 1987). The effects of PLA2‐related metabolites, prostaglandins, leukotrienes and platelet‐activating factor (PAF) were investigated using fluorescein isothiocyanate‐α‐bungarotoxin. Peak and total fluorescence within a cluster were used as indices of clustering and level of n‐AChR, respectively. Prostaglandin E2 (PGE2, 1–10 μm) decreased both indices in a concentration‐dependent manner. Aspirin and indomethacin, cyclooxygenase inhibitors, increased the indices at 1.0 μM and 10–30 nM, and decreased them at higher concentrations of 10–30 μM and 0.1–1 μM, respectively. Prostaglandin F2α (PGF2α, 1–10 μM), nordihydroguaiaretic acid (30 μM), a lipoxygenase inhibitor, and PAF (10 μM) had no effect. These results suggest that the control of endogenous PLA2 on the clustering and level of n‐AChR is due to PGE2, but not to PGF2α, leukotrienes or PAF.
Differentiation of a stem cell line toward a neuronal phenotype
Authors: Cheun Jane E. , Yeh Hermes H.
Published: April 2003

This study examined the morphology and the development of inward currents in the course of differentiation of a stem cell toward a neuronal phenotype. Using the P19 embryonal cell line, whole‐cell current profiles of P19 cells before, during and after retinoic acid‐induced differentiation were matched with their morphology as well as with the expression of neuron‐specific enolase‐like immunoreactivity. Prior to and during the initial 48 hr of retinoic acid treatment, P19 cells either lacked detectable currents or expressed a voltage‐dependent outward potassium current, did not display neuron‐like morphology and did not express neuron‐specific enolase‐like immunoreactivity. Upon completion of retinoic acid treatment, the current profile of fully differentiated P19 cells was hallmarked by a large voltage‐dependent inward current which consisted of a sodium current and a smaller cobalt‐sensitive calcium component, in addition to the potassium current observed earlier. Such cells invariably emitted neuntes and displayed neuron‐specific enolase‐like immunoreactivity. Interestingly, coupling was prevalent among P19 cells in the undifferentiated state but was absent in the fully differentiated cultures. In studying cells undergoing neuronal differentiation, these results underscore the importance of taking into account both electrical properties and morphological considerations in determining the degree of differentiation.
Ontogeny of glycerol phosphate dehydrogenasepositive oligodendrocytes in rat brain. Impaired differentiation of oligodendrocytes in the myelin deficient mutant rat
Authors: Gordon Marcia N. , Kumar Shalini , de los Monteros Araceli Espinosa , de Vellis Jean
Published: April 2003

The ontogeny of oligodendrocytes in the myelin deficient (md) rat mutant and in control rats was explored immunohistochemically using an antiserum against the oligodendrocyte specific enzyme, glycerol phosphate dehydrogenase (GPDH), and the avidin‐biotin complex technique. In control rats, GPDH was demonstrated to be expressed relatively early in oligodendrocyte differentiation, prior to either myelin basic protein or proteolipid protein expression. With development, oligodendrocytes containing GPDH increased in number, apparent staining intensity, cell soma area and process elaboration. Fewer GPDH+oligodendrocytes were observed in the brain of mutant rats than in unaffected littermates at all developmental ages, and major developmental increases in oligodendrocyte density were delayed. The density of GPDH+oligodendrocytes was reduced by about 40% in both the corpus callosum and in the cingulate cortex of P22–25 md mutants compared with control rats. The oligodendrocyte cell soma area was not influenced by the md condition, and increased 2‐fold with development in rats of both genotypes. The area of coronal sections occupied by the corpus callosum increased about 2.5‐fold with development, and was 30% smaller in mutant rats late in their lifespan than in unaffected littermates. The reductions in oligodendrocyte density reported here are of insufficient magnitude to fully account for biochemically measured reductions in oligodendrocyte gene expression accompanying the md trait, indicating that gene expression per oligodendrocyte is also impaired. Cell counts in control rats also revealed that oligodendrocytes are overproduced during development. Cell density and the total number of corpus callosum GPDH+oligodendrocytes per section were maximal at P22–25 and then decreased to adult values. These results suggest that glial cells, like neurons, may be generated in excessive numbers, and some subsequently die, as a normal concomitant of development.
Effects of nerve growth factor and acetyl‐l‐carnitine arginyl amide on the human neuronal line HCN‐1A
Authors: Westlund K.N. , Lu Y. , Werrbach‐Perez K. , Hulsebosch C.E. , Morgan B. , Pizzo D.P. , Eisenberg H.M. , Perez‐Polo J.R.
Published: April 2003

The HCN‐1A clonal cell line, derived from the cortical tissue of a patient with unilateral megencephaly, was shown to differentiate into a mature neuronal‐like state in the presence of the nerve growth factor, dibutyryl cyclic adenosine, 3′,5′‐monophosphate and either 1‐isobutyl‐3‐methylxanthine or forskolin. Differentiation was assessed by measuring the percentage of cells that displayed branched, varicose processes that stained for synaptophysin. Treatment of cultures with a cocktail containing forskolin increased immunocytochemical staining for gamma aminobutyric (GABA), neurofilament protein and the nerve growth factor receptor species p75NGFR. Treatment with acetyl‐l‐carnitine alone had some effects on the cell morphology while acetyl‐l‐carnitine arginyl amide and nerve growth factor together increased the GABA content. Positive staining levels for the neurotransmitters gamma aminobutyric acid, glutamate, somatostatin, cholecystokinin and vasoactive intestinal polypeptide were measured quantitatively for HCN‐1A under basal conditions.
Brain filament proteins in primary cultures derived from chick embryos early in development
Authors: Dahl D. , Maggini L. , Gilad V.H.
Published: April 2003

Brain filament expression and neurofilament post‐translational modifications (phosphorylations) were studied in primary cultures derived from whole 3–4 day chick embryos. After 2–3 days in culture, neurofilament‐positive cells formed neuronal 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. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the flat cells surrounding the neuronal aggregates remained constantly GFAP‐negative throughout the whole experimental period (10 days). GFAP‐positive cells were first observed within the neuronal aggregates on day 8 in vitro.
Calcitonin gene‐related peptide immunoreactive nerve fibers in the developing salivary glands of the rat
Authors: Salo Antero , Törnwall Jyrki , Virta Erkki , Uusitalo Hannu
Published: April 2003

The appearance and distribution of calcitonin gene‐related peptide immunoreactive (CGRP‐IR) nerve fibers was studied by indirect immunofluorescence microscopy in the developing salivary glands of the rat at daily age intervals from the 15th day in utero (i.u.) until birth and subsequently on the 2nd, 5th, 7th, 16th, 30th postnatal (p.n.) days. The findings were compared to samples of adult rats. CGRP‐IR fibers appeared on day 20 i.u. both in parotid and submandibular glands. In submandibular glands, fibers were first seen surrounding the developing ductal branches, in the mesenchyme and also in association with developing ducts and blood vessels. In the parotid gland CGRP‐IR fibers first appeared around ductal branches and blood vessels on day 20 i.u. and on day 21 i.u. CGRP‐IR nerve fibers were found in the mesenchyme and around developing ducts. The density of CGRP‐IR fibers was highest between the 2nd and 16th postnatal days, after which the amount of CGRP immunoreactivity slowly decreased to the levels of adult glands. The increase in a number of CGRP‐IR nerve fibers especially around the ductal and vascular elements in the developing salivary glands indicates that they may play a role in the functional maturation of salivary glands.
Influence of tongue myoblasts on rat dissociated hypoglossal motoneurons in culture
Authors: Ternaux Jean‐Pierre , Portalier Paule
Published: April 2003

Hypoglossal motoneurons of 1–2 day‐old newborn rats were retrogradely labelled following an injection of fluorescent latex microspheres and carbocyanines. Motoneurons were identified among the cell population of the hypoglossal nuclei during the dissociation and culture procedures. DiI labelled motoneurons could be maintained in culture on poly‐l‐lysine coating and Dulbecco Minimum Essential Medium with Ham F12 complement, supplemented with additives and 3% fetal calf serum. Neuronal survival as well as extension of neurites, identified by their content in DiI or by the presence of choline acetyltransferase immunoreactivity, was increased in the presence of myoblastic satellite cells originating from tongue muscular expiants. Co‐cultures of dissociated hypoglossal cells with tongue myoblasts revealed the presence, after 10–15 days in culture, of structures morphologically similar to neuromuscular junctions. Such re‐innervated muscular fibres exhibited muscular contractions which were blocked by curare and augmented by glutamate applications, demonstrating the functionality of the observed re‐innervations.
Mesenteric neurons in the adult rat are responsive to ileal treatment with benzalkonium chloride
Authors: Cracco C. , Filogamo G.
Published: April 2003

The application of a 2 mM solution of the cationic surfactant benzalkonium chloride (BAC) to an ileal segment produced a selective and extensive myenteric denervation. The aim of the present study was to investigate whether such a selective unbalance of the enteric nervous system in the adult rat elicited any plastic response within the mesenteric nervous structures contacting it. Acetylcholinesterase (AChE) staining, β‐nicotinamide adenine dinucleotide (NADH) and β‐nicotinamide adenine dinucleotidephosphate (NADPH) diaphorase histochemistry and glyoxilic acid‐induced fluorescence were performed on whole‐mount preparations of myenteric plexus and mesenteric nerves. In both control and BAC‐treated animals nervous elements were detected along the mesenteric nerves. Although rather similar in position, shape and size, these neurons displayed striking differences with regard to their arrangement and density per nerve. In the controls, few small aggregates of neurons could be detected; more commonly, isolated nerve cell bodies were scattered along the nerve trunks. In the BAC‐treated animals, numerous spherical or spindle‐shaped clusters of neurons were located along the nerves; the mean neuronal density per nerve displayed a two‐five‐fold increase over the control values. The observed changes within the mesenteric nerves might be involved in an attempt at reinnervation of the BAC‐treated intestinal segments from extraenteric sources.
The migration of astrocytes after implantation to immature brains
Authors: Lund R.D. , Zhou H.‐F. , Yee K.T.
Published: April 2003

Using a species‐specific marker, we have found that astrocytes, taken from donors of varying ages from fetal to adult, migrate in highly Stereotypic patterns in immature host brains. Migration is primarily within and towards cell layers, although some cells are seen to migrate along fibre bundles. This contrasts with studies using the same approach in mature hosts, where migration is predominantly within fibre layers, largely excluding cellular regions.
Reactive astrocytes in explant cultures of goal scars derived from lesioned rat optic nerve: An ultrastructural study
Authors: Trimmer Patricia A.
Published: April 2003

Explant cultures of glial scars generated by surgical removal of the retina in 3–60‐day‐old rats were used to determine if reactive astrocytes survive in vitro and how closely reactive astrocytes in culture resemble their in vivo counterparts. Characterization of the composition of age matched glial scars in vivo and in vitro showed that reactive astrocytes survived in glial scar explants even after several weeks in culture. Reactive astrocytes in both neonatal and adult glial scars retained ultrastructural features characteristic of reactive astrocytes in vivo. However, fewer reactive astrocytes survived in culture when explants were prepared from adult rat glial scars. The results of this study demonstrate that tissue culture is a viable model for the study of reactive astrocytes. A critical factor in the survival of reactive astrocytes in culture was the complete removal of myelin debris prior to the establishment of the culture. This outcome suggests that it will be important to clarify why myelin debris persists in culture and how it affects the survival of reactive astrocytes.
Immunohistochemical localization of basic fibroblast growth factor in cultured rat astrocytes and oligodendrocytes
Authors: Vijayan V.K. , Lee Y.L. , Eng L.F.
Published: April 2003

The distribution of basic fibroblast growth factor in cultured astrocytes and oligodendrocytes was examined using immunocytochemistry. The results demonstrate a localization of basic fibroblast growth factor immunoreactivity predominantly in astrocyte nuclei at all stages of differentiation. Cytoplasmic and process staining was best detected during early stages of differentiation, under normal growth conditions or as a result of treatment with dibutyryl cyclic adenosine monophosphate. Astrocytes at all stages of differentiation bound antibody‐complexed bFGF, suggesting the presence of cell‐associated low affinity binding sites for the growth factor. Our studies also show the presence of immunoreactivity for basic fibroblast growth factor in process‐bearing oligodendrocytes. These results suggest a role for endogenous basic fibroblast growth factor in astrocyte and oligodendrocyte growth and function.
The neurotrophic peptide Org 2766 does not influence the expression of the immediate early gene c‐fos following sciatic nerve crush in the rat
Authors: Plantinga L.C. , Verhaagen J. , Wong S.L. , Edwards P.M. , Bär P.R. , Gispen W.H.
Published: April 2003

The neurotrophic peptide Org 2766 accelerates the regeneration of peripheral nerves. Although the mechanism of action of this neuropeptide is not yet understood, functional, pharmacological, and morphological evidence has demonstrated that Org 2766 exerts its beneficial effect during the early stages of nerve regeneration. The induction of some members of the Immediate Early Gene (IEG) family such as c‐jun and c‐fos is one of the first molecular events following peripheral nerve damage. The Fos and Jun proteins act as a transcription factor and may stimulate the expression of a number of genes implicated in nerve regeneration. We examined whether Org 2766 stimulates nerve regeneration by enhancing or prolonging the expression of c‐fos mRNA. Following a crush lesion of the sciatic nerve, the expression of c‐fos mRNA was induced in the spinal cord and in the damaged nerve at 30 min following injury in untreated animals as demonstrated with Northern blot. No effect of the crush lesion was observed in dorsal root ganglia (DRG). The induction of c‐fos mRNA in the damaged nerve was more robust as compared to the relatively small induction observed in the spinal cord. With in situ hybridization an increase in c‐fos mRNA expression both in the dorsal and in the ventral horn of the spinal cord was demonstrated at 30 min post‐lesion. In the distal sciatic nerve portion the expression of c‐fos mRNA was predominantly localized around Schwann cell nuclei at 30 min after nerve crush.

The effect of Org 2766 treatment on the expression of c‐fos mRNA was investigated using semiquantitative dot blots. Dot blots containing RN A samples of DRG, spinal cord or sciatic nerve of control animals, saline treated and Org 2766 treated animals isolated 15 min, 30 min, l hr, 1 day and 2 days following nerve crush were scanned densitometrically. No significant effect of Org 2766 on the expression of c‐fos mRNA was detected. We conclude that Org 2766 does not stimulate peripheral nerve regeneration by affecting the expression of the c‐fos mRNA following sciatic nerve crush. The putative effect of Org 2766 on other immediate early transcription factors of the fos and jun family is currently under investigation.
Altered vasoactive intestinal polypeptide gene expression in the fetal rat suprachiasmatic nucleus following prenatal serotonin deficiency
Authors: Ugrumov M.V. , Trembleau A. , Calas A.
Published: April 2003

This study has evaluated the possible role of serotonin, a potential morphogen, in the regulation of vasoactive intestinal polypeptide (VIP) gene expression in the target neurons of the suprachiasmatic nucleus (SCN) before and after the onset of the serotonin neurotransmitter function. VIP gene expression was quantified by in situ hybridization of the corresponding mRNA on cryostat sections with subsequent film autoradiography and densitometry. The content of VIP mRNA was measured in the SCN in fetuses at the 21st embryonic day (E21) and in postnatal rats at day 11 (P11) following chronic depletion of serotonin by p‐chlorophenylalanine, an inhibitor of serotonin synthesis. This inhibitor was daily injected to pregnant rats for E13–20 or to postnatal animals for P2–10. Results of this study indicate that prenatal serotonin depletion caused a significant increase in VIP mRNA content in the SCN compared to control fetuses. On the contrary, the same treatment performed postnatally did not change VIP mRNA levels in the SCN. These data suggest that the VIP gene expression in differentiating target neurons of the SCN might be under serotonin inhibitory control during prenatal neurogenesis, prior to the onset of the serotoninergic neurotransmission.