bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of exogenous gangliosides on neurons in culture: A morphometric analysis
Authors: Massarelli R. , Ferret B. , Gorio A. , Durand M. , Dreyfus H.
Published: April 2003

Cultures of isolated neurons have been treated with a purified preparation of gangliosides (10−5M and 10−9M) added to the cell growth medium at the 3rd day in culture and a morphometric analysis of the cells was performed with an image analyzer after 1 and 4 days of treatment. The number of cells and the area of the cell bodies were increased following the treatment. The results indicate as well the ‘sprouting’ effect of the glycolipids on the number of secondary neuronal processes and an increase in the length of the primary neuntes. The present data and other biochemical evidence (Dreyfus et al., 1984, J. Neurosci. Res.) suggest that the addition of exogenous gangliosides may have a trophic effect on neurons, greatly enhances the number of cell to cell contacts, and, possibly, stimulates cell proliferation and differentiation.
Pheochromocytoma PC12 cells influence the differentiation of co‐cultured skeletal myotubes
Authors: Defez Roberto , Brachet Philippe
Published: April 2003

Rat skeletal myotubes were cultured in the presence or absence of pheochromocytoma PC12 cells under experimental conditions permitting the maintenance of the muscle cells for up to 5 weeks. The number of acetylcholine receptors (AChRs), estimated using radioiodinated α‐bungarotoxin (α‐BTX), was comparable in both types of culture for the first 2 weeks. After prolonged incubation (4 weeks), however, α‐BTX binding sites were two times more abundant in the PC12‐myotube cultures than in the myotube cultures. This is likely to reflect a higher number of AChRs at the surface of the myotubes, for control experiments showed that the contribution of PC12 cells was negligible. Presence of PC12 cells also increased the number of AChR aggregates. Use of fluorescent α‐BTX demonstrated that mixed cultures contained two times more receptor patches than did myotubes alone during the first 2 weeks of culture, and up to five times more after 4 weeks. Essentially all the tight contacts established between PC12 cells and myotubes were characterized by a receptor cluster and, in some cases, by an aggregate of an acetylcholine esterase (AChE), as visualized by immunohistochemistry. Furthermore an antiserum specific for the adult (fast) isoform of the myosin heavy chain stained some myotubes in longterm, mixed cultures. The antigen was never observed in sister cultures of myotubes alone. These data show that PC12 cells, which are thought to derive from the medullary part of the adrenal gland, can influence the differentiation of skeletal muscle cells in several ways.
Multiple forms of aryl acylamidase in regional tissues of developing rat brain
Authors: Hsu Louise L.
Published: April 2003

The specific activities of two forms of aryl acylamidase (AAA) were examined in 7 regions of the developing rat brain, plus the remainder of the brain and the whole brain. AAA‐1 activity peaked at 15 days old in all brain regions studied except the whole brain where it peaked at 22 days of age. AAA‐2 activity peaked between 15 and 29 days old in most brain regions studied except corpus striatum and hippocampus where the AAA‐2 activity peaked before 15 days old. In all areas and at all time periods, with the exception of CS after 15 days of age, AAA‐2 activity was much higher than that of AAA‐1. The developmental pattern of AAA‐1 is generally the same in the different brain regions while that of AAA‐2 shows more regional specificity. These results indicate that neither AAA‐1 nor AAA‐2 may be associated with amine N‐acetyltransferase in the brain which has an entirely different developmental pattern.
The binding properties and regional ontogeny for [3H]glutamic acid Na+‐independent and [3H]kainic acid binding sites in chick brain
Authors: Voukelatou G. , Angelatoy F. , Kouvelas E.D.
Published: April 2003

The binding kinetics, pharmacological properties and regional ontogeny of l‐[3H]glutamic acid Na+‐independent and [3H]kainic acid binding sites were studied in preparations of chick brain. One binding component was found for l‐[3H]glutamic acid with a Kd value of 176 × 10 9M. For [3H]kainic acid two binding components were found in the hemispheres, optic lobes and brain stem, one with high affinity and a Kd value of 12.5 × 10 9M and one with low affinity and a Kd value of 260 × 10 9M. In cerebellum only one binding site was detected for [3H]kainic acid with a Kd value of 144 × 10 9M. The ontogeny of l‐[3H]glutamic acid and [3H]kainic acid binding sites was studied using membrane preparations (48,000 g pellet) of hemispheres, optic lobes, brain stem and cerebellum. Binding of l‐[3H]glutamic acid was already significant in all brain regions by embryonic day 11 but major increases in total receptor number per brain region or per mg of protein were apparent by embryonic day 19 and especially after hatching. Cerebral hemispheres, optic lobes and brain stem showed few [3H]kainic acid binding sites by day 13 in ovo. An increase follows which, in hemispheres and optic lobes, continues at the same rate during the first two weeks after hatching. In cerebellum, by contrast, the kainic acid binding site is almost undetectable until embryonic day 15. The appearance of these binding sites in cerebellum takes place during the restricted period between days 15 in ovo and 5 post‐hatching. This pattern of development of [3H]kainic acid binding sites almost parallels the developmental patterns of the molecular layer of chick cerebellum and it is consistent with the results of our autoradiographic study showing that the great majority of kainic acid binding sites are localized in the molecular layer.
Fetal ethanol exposure: A morphometric analysis of myelination in the optic nerve
Authors: Samorajski T. , Lancaster F. , Wiggins R. C.
Published: April 2003

Pregnant Long‐Evans rats were fed a liquid diet containing ethanol during gestation. Controls consisted of both pair‐fed dams and dams fed ad libitum with an equivalent, iso‐caloric diet lacking ethanol. Subsequent effects of ethanol measured in the offspring include a significant lag in the rate at which non‐myelinated axons are lost in association with the initial overproduction of neurons. Additionally, there was a slight lag in the rate of acquisition of myelinated axons; and altogether there was a large increase in the ratio of non‐myelinated to myelinated axons. Frequency spectra of myelinated and non‐myelinated axons by size were normal, and the relationship between axon size and myelin lamellae was also normal. Measured against the dynamic, normal background of rapid cell‐loss and the progressive development of myelin, morphometric demonstration and evaluation of the comparatively small divergences associated with fetal alcohol exposure are difficult; nevertheless, these results are consistent with and help account for the marginal hypomyelination previously observed by quantitative neurochemistry.
Polyamines affect growth of cultured rat cerebellar neurons in different sera
Authors: Gilad Gad M. , Gilad Varda H.
Published: April 2003

The study examines the effects of polyamines on growth of cultured neurons from 6‐day‐old rat cerebellar cortex, by means of: (a) irreversible inhibition of ornithine decarboxylase activity with α‐difluoromethylornithine, and (b) treatment with the exogenous diamine putrescine and the polyamines spermidine and spermine, in the presence of sera from different sources. Inhibition of ornithine decarboxylase activity starting at plating time led after 24 hr to a partial inhibition of cell aggregation with a drastic (90%) inhibition of neurite formation. However, after 48 hr of enzyme inhibition aggregation and neurite formation increased to approach the 24 hr control values and eventually cultures fully recovered. Polyamines added at plating time in the presence of fetal calf serum led to a permanent dose‐dependent inhibition of aggregation and neurite formation, spermine being effective at lower doses (spermine < spermidine ⪡ putrescine). Adhesion of cells to polylysine coated surfaces was not affected by polyamines. Recovery from the cytostatic polyamine effect was observed after washing and addition of fresh medium. Prevention of the effect was achieved in the presence of aminoguanidine, an inhibitor of diamine and polyamine oxidases. The preventive effect of aminoguanidine was dose polyamine‐dependent, with higher aminoguanidine concentrations needed to prevent the spermine effect (spermine > spermidine > putrescine). The polyamine effects were observed in the presence of fetal calf, heat‐inactivated fetal calf and human sera, but not with rat serum. Addition of polyamines to 24‐hr‐old cultured neurons, in the presence of fetal calf serum, led 12 hr later to cell death. This lethal effect could be inhibited by aminoguanidine. We conclude: (a) irreversible inhibition of ornithine decarboxylase activity delays but does not prevent neuronal growth in culture; (b) oxidation products of extracellular polyamines inhibit cell aggregation and neurite formation of cultured neuroblasts, and have lethal effects on growing neurons in culture, and (c) different pharmacological effects of polyamines can be expected in different species.
Histochemical distribution of acid glycosaminoglycans in rat cerebellum during postnatal development: Neuropile
Authors: Alvarado M.V. , Castejon H.V.
Published: April 2003

Developmentally regulated modifications of glycosaminoglycans (GAGs) in the central nervous system (CNS) have suggested that also in the CNS, these compounds might participate in morphogenesis and nerve cell differentiation. However very few studies have been reported concerning the regional distribution of these compounds by histochemical techniques. We have used the Alcian Blue staining method also in conjunction with enzymatic digestion and with a technique which allowed the measurement of the degree of GAG sulphatation. The combination of the three techniques showed that during the first week GAGs, presumed to be hyaluronic acid, are localized throughout the neuropile of the entire cerebellum and especially in the medullary region. Sulphated glycans appear later in the medullary region (particularly at the border between the medullary region and the internal granular layer) and in all the layers of cerebellum (in particular around the Purkinje and deep cerebellar nuclei neurons and possibly in the cerebellar glomeruli). Sulphated glycans in the medullary region disappear around the 12th day when myelination starts. The transient presence of glycoproteins in the molecular layer was also detected.
Benzodiazepine and muscarinic cholinergic binding sites in striatum and brainstem of the human fetus
Authors: Schlumpf M. , Lichtensteiger W.
Published: April 2003

The regional distribution of binding sites for [3H]flunitrazepam and [3H]N‐methyl‐scopolamine was studied in human fetal brain of 18 and 23 weeks gestation by in vitro autoradiography. Areas densely labelled by both ligands include the hypoglossal nucleus and the striatum. The latter is characterized by a patchy distribution of both binding sites reminiscent of acetylcholinesterase staining. Similarities with the rat fetus and the occurrence of binding sites in areas of potential significance for fetal behaviour are discussed.
Procedure for establishing oligodendroglial cells in primary cultures based on developmental parameters
Authors: Juurlink B. H. J. , Devon R. M.
Published: April 2003

We have established cultures using dissociated cells obtained from the neopallium of mouse pups at several different stages of development. The cellular composition of these cultures changed from more than 95% astrocytes when cultures were established from the neopallium of neonates to more than 90% oligodendrocytes when cultures were established from the neopallium of 2‐week‐old pups. The oligodendrocytic nature of the cells was established on morphological bases as well as the presence of an oligodendrocytic marker, galactocerebroside.
Dysmyelinogenesis in caprine β‐mannosidosis: Ultrastructural and morphometric studies in fetal optic nerve
Authors: Lovell Kathryn L. , Boyer Philip J.
Published: April 2003

The optic nerves from a goat fetus affected with β‐mannosidosis and a control fetus were analysed morphologically in order to investigate developmental aspects of β‐mannosidosis‐associated myelin deficits. In the affected fetus, the number of myelinated axons per unit area was about 25% of the control values. Histograms of axonal diameter indicated that a greater percentage of the myelinated and unmyelinated axons were of larger caliber in the affected fetus than in the control fetus and that very few small axons were myelinated in the affected animal. The mean values of myelin sheath thickness in the affected and control animals did not differ significantly. Ultrastructural analysis revealed a decreased proportion of oligodendrocytes and an increased proportion of astrocytes in the affected fetus.

These results indicate that the pathogenetic process leading to cellular abnormalities and myelin deficits in β‐mannosidosis has been initiated prior to 124 days gestation, during an early stage of myelination in the goat optic nerve. The decrease in number of oligodendrocytes suggests that early cell death and/or change in oligodendrocyte proliferation contribute to the myelin deficit. Analysis of the prenatal development of lesions will help clarify the pathogenesis of dysmyelinogenesis in β‐mannosidosis.
Catalase protection of neuronal survival in vitro is not directed to the accumulation of peroxides in the culture medium
Authors: Martin Estralita M. E. , Skaper Stephen D. , Varon Silvio
Published: April 2003

Walicke et al. (1986, J. Neurosci. 6, 1114–1121) have shown that catalase can replace the pyruvate requirement for survival of CNS neurons cultured in vitro. Since presently the only known function of catalase is the enzymatic degradation of hydrogen peroxide to water and oxygen, the simplest interpretation of the ability of catalase to support neuronal survival would be that catalase removes from the culture medium hydrogen peroxide. To test this hypothesis 8‐day embryonic chick forebrain cells were cultured for 24 hr in a modified Eagle's Basal Medium with the serum‐free supplement N1 (HEBM/N1) in the presence or absence of Phenol Red, 20 μg/ml catalase, 1 mM pyruvate, and/or 25 mM N‐2‐hydroxyethylpiperazine‐N′‐2‐ethane‐sulfonic acid (HEPES) on a polyornithinelaminin substratum. The various media were then assayed for peroxide content using the potassium iodide method described by Wang and Nixon (1978, In Vitro14, 714–722). The present data reveal that (1) HEBM/N1 normally contains approximately 50 μM peroxides, little of which is hydrogen peroxide, (2) the organic peroxide levels accumulating in this medium are not reduced by either catalase or pyruvate, and (3) medium modifications can reduce to no longer detectable levels the peroxides accumulating in the medium, but catalase or pyruvate is still required for neuronal survival. We conclude that catalase must exert its survival‐promoting action at levels other than peroxides accumulating in the culture medium.
Axonal maturation in development—II. Immunofluorescence study of rat spinal cord and cerebellum with axon‐specific neurofilament antibodies
Authors: Bignami A. , Dahl D.
Published: April 2003

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon‐specific anti‐bodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post‐translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon‐specific antibodies first appeared in motor and sensory axons at different times in development: day 13–14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2(1 monoclonal). There were no major differences between conventional and axon‐specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post‐translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.
The trophic responses of avian sensory ganglia in vitro to N‐acetylated and des‐acetyl forms of α‐melanocyte stimulating hormone (α‐MSH) are qualitatively distinct
Authors: Haynes Laurence W. , Semenenko Frances M.
Published: April 2003

α‐Melanocyte‐stimulating hormone (α‐MSH) accelerates the regrowth of peripheral nerve axons in the rat following their transection (Verhaagen et al., Expl Neurol. 92, 451–454, 1986). The cellular mechanisms of this trophic response were investigated for several naturally occurring derivatives of α‐MSH using Nerve Growth Factor (NGF)‐stimulated quail sensory ganglion explants in vitro in which both neurite outgrowth and non‐neuronal cell behaviour could be more reliably observed and quantified. Neurite outgrowth was determined with a semi‐quantitative scoring assay. Glial migration into the outgrowth was quantified using a monoclonal antibody, GTE‐52, which labels the nuclei of Schwann cells. Des‐acetyl α‐MSH caused a marginal increase in the neurite outgrowth density which was significant at concentrations of 0.04 and 0.1 μg/ml. The response to acetylated (N‐acetyl, N,O‐diacetyl) forms of α‐MSH was characterized by fascicle formation by neurites which resulted in an apparent decrease in the neurite score and by the outgrowth of non‐neuronal cells. Using monoclonal antibody GTE‐52, which recognizes a glial nuclear antigen, these cells were identified as Schwann cells. N‐Acetyl, but not des‐acetyl α‐MSH increased the number of GTE‐52‐labelled cells in the NGF‐stimulated neurite outgrowth and stimulated their migration in the absence of neurites when NGF was omitted from the culture medium. Exposure of growing explants to two polyclonal antibodies against α‐MSH resulted in an increased neurite outgrowth density. The results support the hypothesis that α‐MSH peptides stimulate peripheral nerve growth by modulating the neurite sprouting response and demonstrate that the nature of the neurotrophic response to naturally occurring melanotropins depends on the existence of acyl substitution at the N‐terminal amino acid residue. A possible role of endogenous melanotropin peptides in the regulation of sensory nerve growth is discussed.
Development and distribution of serotonin in the central nervous system of Manduca sexta during embryogenesis I. The brain and frontal ganglion
Authors: Radwan Wafaa A. , Granger Noelle A. , Lauder Jean M.
Published: April 2003

Development of the serotonergic system in the brain and frontal ganglion of the Manduca embryo between 35 and 100% of development was studied immunocytochemically with an antiserum to serotonin (5‐HT). Serotonin immunoreactivity was initially detectable at 40–45% development in short fibers in the head region, prior to differentiation of the brain. Immunoreactive cell bodies were first seen in the brain at 60% development, located in the protocerebrum and tritocerebrum. Thick fiber tracts crossing the midline (commissures) could also be observed at this early stage. As development of the embryo progressed, eight immunoreactive cell groups, containing a total of about 38–40 cells and four commissures with terminal arborizations appeared successively in the brain. From 75 to 100% development, no obvious changes occurred in the number or distribution of cells and the brain exhibited the same pattern of 5‐HT immunoreactive cells, fiber tracts and arborizations as in last instar larvae of Manduca. However, an increase in the size of the cells in both the brain and frontal ganglion was noted between 75 and 80% development, followed by a decrease by 100% development. The frontal ganglion was found to contain three 5‐HT immunoreactive cells, which appeared to send bilateral projections into the frontal connectives and the recurrent nerve. During embryonic development, the dendritic arborizations of these frontal ganglion cells increased, while the amount of 5‐HT immunoreactivity in the cell bodies decreased. Thus, the serotonergic system first appears in the Manduca embryo at an early stage of development, similar to the situation in other insects as well as vertebrates. By the end of the embryonic period, the same number of serotonergic neurons are present in the brain as in larval and adult Manduca, suggesting that once formed, these cells persist through postembryonic development and metamorphosis.
In vitro screening for anticonvulsant‐induced teratogenesis in neural primary cultures and cell lines
Authors: Regan C. M. , Gorman A. M. C. , Larsson O. M. , Maguire C. , Martin M. L. , Schousboe A. , Williams D. C.
Published: April 2003

To establish inherent potential for the induction of neural tube defects the ability of selected anticonvulsant agents to interfere with cell division has been established in vitro using an antiproliferative assay in clonal cell lines and a cytotoxicity assay using primary cultures of cerebral cortex neurons at different stages of development. In order to evaluate the relative toxicities of these agents their in vitro effects were determined at 2–3 times the plasma therapeutic level. By these procedures valproate and the benzodiazepines, diazepam and clonazepam, exerted a potent antiproliferative action which could not be attributed to increased cytotoxicity. In contrast phenytoin was markedly cytotoxic but was without an antiproliferative action. This cytotoxicity was most pronounced during the periods of extensive fibre outgrowth. When compared to epidemiological and animal study data, agents which inhibited cell proliferation within twice therapeutic concentration were consistently associated with major neural tube malformations. However phenytoin, found to be positive in the cell cytotoxicity assay, is not associated with neural tube malformations but rather is primarily associated with mental retardation. Thus assessment of antiproliferative activity of anticonvulsant drugs may be one criterion for identification of teratogenic potential during neurulation.
Immunocytochemical study of S‐100 positive glial cells in the brainstem and spinal cord of the rat embryo
Authors: Gomez Luciano Ariel , Brusco Alicia , Saavedra Jorge Pecci
Published: April 2003

A light and electronmicroscopic immunocytochemical study of the glial cells in the brainstem and spinal cord of the 18th day rat embryo was performed using an anti‐S‐100 protein antiserum. Only the radial glia and the free immature glial cells are S‐100 immunoreactive. Neurons are devoid of S‐100 immunoreactivity. The radial glia form two paramedial plates and a great number of lateral plates, uniformly spaced along the ventral portion of the brainstem from the mesencephalon to the medulla. The S‐100 protein was also detected in the perivascular membranes and glial limitans. Embryonic glia adopt a highly organized spatial pattern in the brainstem that could set the structural basis for an organized assembly of the developing nervous tissue. The use of the S‐100 protein as a glial marker in the embryonic rat brain proved to be of great value. Antibodies to S‐100 protein allow the demonstration of immature glial cells and a highly organized spatial pattern in the brainstem and spinal cord of the rat embryo.
Cerebral ornithine decarboxylase levels following gestational exposure to cocaine
Authors: Bondy S. C. , Nakla M. , Ali S. F. , Ahmad G.
Published: April 2003

The pre‐ and postnatal developmental course of cerebral ornithine decarboxylase (ODC) has been studied in infant rats after treatment of pregnant dams with cocaine. Levels of cocaine attained in brains and serum of embryos were not initially increased over corresponding maternal values, but were more persistent. However, cocaine was no longer detectable in these tissues 4 days after administration. The cerebral ODC level of treated pups was initially depressed and subsequently elevated relative to control values. These changes were apparent at times when cocaine was not detected in the developing brain. Results indicate that a transient exposure to cocaine in utero may lead to prolonged developmental abnormality.
Motor nerve terminal restoration after focal destruction in young and old mice
Authors: Robbins Norman , Kuchynski Marie , Polak Joseph , Grasso Alfonso
Published: April 2003

Regeneration of soleus motor nerve terminals after focal destruction by black widow spider venom (BWSV) or its active factor alpha‐latrotoxin (LTx) was compared in young and old CBF‐1 mice. The object was to determine whether previously reported delayed regeneration after nerve injury in old rodents was due to altered removal of debris, or delay or aberrancy in structural or functional restoration of the neuromuscular junction. In addition, the use of a new fluorescent technique permitted for the first time quantitation of the accuracy of early nerve terminal regeneration in mammalian muscle. Immunohistochemical and electron micrographic studies showed no age difference in destruction of terminals and removal of debris 2 days after toxin application. The indirect twitch and structural reinnervation (measured with flourescent techniques) returned to an equal extent in young and old mice beginning at 3 days after LTx treatment. BWSV (as opposed to LTx) delayed regeneration 1 day in young but not in old mice. On the first day of reinnervation, there was perisynaptic outgrowth in both young and old mice, although in the latter, there was a higher incidence of aberrant outgrowth. The relation between return of twitch strength and recovery of nerve terminal area (measured in teased zinc iodide‐stained preparations) showed no age dependency. We conclude that factors cited to explain altered reactive sprouting in the aging CNS do not apply to regeneration of peripheral motor nerve terminals. However, it is possible that the aberrant regrowth observed at the neuromuscular junction in old mice will pertain to the aging CNS. Altered axonal rather than nerve terminal regeneration is the likely source of delayed peripheral nerve regeneration in old animals.
Aging and stress‐induced changes in choline and glutamate uptake in hippocampus and septum of two rat strains differing in longevity and reactivity to stressors
Authors: Gilad Gad M. , Gilad Varda H. , Tizabi Yousef
Published: April 2003

Stress induced changes in neurochemical indices of neurotransmission are more pronounced in the septohippocampal cholinergic system of Wistar Kyoto rats, which are behaviorally more reactive to Stressors and have a shorter life span, than in Brown Norway rats. Moreover, pronounced degeneration of septohippocampal cholinergic neurons occurs earlier in life in Wistar Kyoto rats. In the present study the high affinity synaptosomal uptakes of choline and glutamate were used as indices for cholinergic and glutamatergic systems respectively. Following 2 hr of mild restraint stress increases in both uptake systems were observed in all regions examined (hippocampus, septum and frontal cortex). The stress‐induced increases were generally similar in young (3 months) and aged (20 months) rats of both strains. The noted exception was that choline uptake levels, which were reduced in the hippocampus of unhandled aged WKY rats, remained unchanged after stress. The results confirm the involvement of the septohippocampal cholinergic system in the response to acute stress and extend the findings to include the hippocamposeptal glutamatergic system activation as well. It is suggested that in spite of neuronal degeneration during aging, these responses to stress can be maintained by compensatory efforts of neurons that remain intact.
Influence of development and aging on nicotinic receptor subtypes in rodent brain
Authors: Zhang Xiao , Wahlström Göran , Nordberg Agneta
Published: April 2003

The influence of development and aging on nicotinic receptor subtypes in rodent brain was investigated. 3H‐nicotine and 3H‐acetylcholine (3H‐ACh) were used as receptor ligands. Specific binding sites for 3H‐nicotine and 3H‐ACh were detected in mouse brain during the late prenatal period. A drop in the number of 3H‐nicotine and 3H‐ACh binding sites was measured shortly after birth. The 3H‐nicotine and 3H‐ACh binding sites showed different time courses during prenatal development. Competition experiments using unlabelled (−)nicotine and3H‐nicotine revealed one population of high affinity nicotinic binding sites in the cortex of 1‐day and 5‐day‐old mice whereas both a set of high and low affinity binding sites were found in adult mice. The proportion of cortical high and low affinity nicotinic binding sites did not change with aging although the absolute amount of high affinity nicotinic binding sites decreased. The 3H‐nicotine binding showed different temperature dependence in rat brain of different ages. The results illustrate dynamic changes in nicotinic receptor properties during life span of rodents.
Influence of early chronic phenobarbital treatment on cerebral arteriovenous differences of glucose and ketone bodies in the developing rat
Authors: Schroeder Henri , Bomont Laurent , Nehlig Astrid
Published: April 2003

The influence of an early chronic phenobarbital treatment on cerebral arteriovenous differences of glucose, lactate, pyruvate, β‐hydroxybutyrate and acetoacetate was studied in suckling rats. The animals were treated from day 2 to 21 after birth by a daily injection of 50 mg/kg phenobarbital or by saline and were studied at 10, 14 and 21 days. Phenobarbital treatment induced a decrease in cerebral arteriovenous difference of glucose at P14 and no change at P10 and P21. The barbiturate did not have any influence on cerebral arteriovenous difference of lactate and pyruvate at the three stages studied. Cerebral uptake of β‐hydroxybutyrate was unchanged at P10 and increased by two‐fold at P14 and by threefold at P21 by phenobarbital. Cerebral arteriovenous difference of acetoacetate was low and did not change with the pharmacological treatment. At P14 and P21, the calculated amount of oxygen used by the brain for the oxidation of ketone bodies was twice as high in barbiturate‐ as in saline‐treated rats and reached values of 47 and 16% respectively in phenobarbital‐exposed animals. In addition, the barbiturate seemed to affect the carrier process of β‐hydroxybutyrate from blood to brain. The results of the present study are in good agreement with previous data from our laboratory showing that an early chronic phenobarbital treatment is able to induce a shift in the cerebral energy metabolism balance in favor of ketone bodies.
Connections of the insular cortex in kittens: An anatomical demonstration with wheatgerm agglutinin conjugated to horseradish peroxidase technique
Authors: Shimizu Hideaki , Norita Masao
Published: April 2003

The postnatal development of the afferent and efferent connections of the feline insular cortex was investigated using wheat germ agglutinin conjugated to horseradish peroxidase method. The overall pattern of connections was found to be substantially the same as that observed in adults. In the cortex, anterograde and retrograde labeling was found in the presylvian sulcus, cingulate gyms, cruciate sulcus, medial prefrontal area, lateral suprasylvian cortex, and posterior rhinal sulcus. Subcortical regions containing label included the lateralis medialis‐suprageniculate nuclear complex, ventral medial thalamic nucleus, claustrum, lateral amygdaloid nucleus, hypothalamus, and raphe nuclei. In addition, axonal labeling was seen in the striatum, superior colliculus, and pontine nuclei.

In contrast, the insular associational projections in kittens differed markedly from those found in adults. In the early postnatal period (1–14 days), dense terminal labeling was found in the cortical layers I, V and VI as well as in the underlying white matter, whereas only moderate to sparse labeling was observed in layers II, III and IV. By four‐weeks, an adult‐like distribution of terminals was present in each cortical areas: labeling was found mainly in layers I, II, III and IV, with less in layers V and VI.

The present results suggest that the basic framework of the insular connections is formed prenatally and that the fine tuning of the axonal terminals and the formation of synapses occurs mainly during the first four postnatal weeks.
Effect of acetyl‐L‐carnitine on forebrain cholinergic neurons of developing rats
Authors: De Simone R. , Ramacci M.T. , Aloe L.
Published: April 2003

It has been shown that the endogenous compound, acetyl‐L‐carnitine (ALCAR), acts in the brain as a metabolic cofactor in the synthesis of acetylcholine. In these studies, ALCAR was injected into the brain of developing rats every other day for the first three weeks after birth in order to assess its effect on forebrain cholinergic neurons. The results showed that intracerebroventricular (icv) administration of ALCAR causes an increase of choline acetyltransferase (ChAT) activity and of nerve growth factor receptor expression in the striatum. Biological assays of brain tissues revealed that the level of nerve growth factor (NGF) in the hippocampus also increases. The ability of brain cholinergic tissues to respond to exogenous administration of ALCAR is discussed.
Insulin receptor in mouse neuroblastoma cell line N18TG2: Binding properties and visualization with colloidal gold
Authors: Sartori C. , Stefanini S. , Bernardo A. , Augusti‐Tocco G.
Published: April 2003

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335–347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system.

We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20°C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligandreceptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects.

On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.
Culture of dorsal root ganglion neurons from aged rats: Effects of acetyl‐l‐carnitine and NGF
Authors: Manfridi A. , Forloni G.L. , Arrigoni‐Martelli E. , Mancia M.
Published: April 2003

In vitro neuronal preparations are used to study the action mechanism of substances which are active in normal and pathological brain aging. One major concern with in vitro assays is that the use of embryonic or adult neurons may hamper an appreciation of the relevance of these substances on aged nervous tissue. In the present study for the first time cultures of aged dorsal root ganglia from 24‐months‐old rats were maintained in vitro up to 2 weeks. This model was used to investigate the neurotrophic/ neuroprotective action of nerve growth factor and acetyl‐l‐carnitine. A large population of aged dorsal root ganglia neurons was responsive to nerve growth factor (100 ng/ml). Nerve growth factor induced an increase of initial rate of axonal regeneration and influenced the survival time of these neurons. Acetyl‐l‐carnitine (250 μM) did not affect the axonal regeneration but substantially attenuated the rate of neuronal mortality. A significant difference was evident between the acetyl‐l‐carnitine‐treated and the untreated neurons from the first cell counting (day 3 in culture). After 2 weeks the number of aged neurons treated with acetyl‐l‐carnitine was almost double that of the controls. The effects of acetyl‐l‐carnitine on aged DRG neurons potentially explain the positive effects in clinical and in vivo experimental studies.
The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary
Authors: Gary Keith A. , Chronwall Bibie M.
Published: April 2003

The onset of dopaminergic innervation and its effects on melanotrope proliferation were investigated in the rat pituitary intermediate lobe. Dopamine, and its synthetic rate‐limiting enzyme tyrosine hydroxylase, were first detected immunohistochemically on late post‐natal day 3 or early postnatal day 4. Axon density was highest at the neural lobe/intermediate lobe border, and decreased toward the pituitary cleft. By post‐natal day 10, the adult pattern of tyrosine hydroxylase immunoreactivity was established and remained through post‐natal day 14. Neurointermediate lobe dopamine levels, measured by HPLC, correlated well with the increased axon density observed in the immunohistochemical studies. Dopamine could not be measured by our assay (100 fg limit) until post‐natal day 3 (439.32 fg/NIL). Dopamine concentration increased to 2.09 ± 0.425 ng at PN 4, 86.31 ± 20.42 ng at PN 7, 168.72 ± 18.37 ng at PN 10. Melanotrope proliferation was determined by [3H]thymidine incorporation before and after innervation. Concomitant with the onset of innervation, the proliferation index dropped from 13.4 ± 0.01% to 6.5 ± 0.002% at PN 4, and continued to decrease until a level of 3 ± 0.003% was established by PN 10. To confirm the inhibitory action of dopaminergic innervation on melanotrope proliferation, rat neonates were injected intracisternally with 150 mg 6‐hydroxydopamine to destroy dopaminergic axons within the intermediate lobe. Measurement of dopamine concentrations in neurointermediate lobes of injected animals showed a decrease in dopamine levels as compared to controls. From PN 4 (0.88 ± 0.165 ng), DA levels gradually increased during development: at PN 5, [DA] = 0.689 ± 0.104 ng; PN 6 [DA] = 11.60 ± 2.24 ng; PN 7 [DA] = 20.93 + 3.80 ng; and PN 10 [DA] = 27.95 ± 3.46 ng. Melanotrope proliferation also increased in 6‐hydroxydopamine‐treated animals. At PN 4, the onset of innervation reduced the pre‐innervation proliferation index to 8.75 ± 0.002%, only a 30% reduction in contrast to the > 50% decrease observed in control animals. A stable proliferation level of approximately 7.5% persisted in all subsequent stages with 6‐OHDA administration.

Our results demonstrated the time of dopamine innervation onset and a characteristic developmental pattern for axons within the rat intermediate lobe. The onset of innervation and increased dopamine concentration suggests increased dopaminergic control of the melanotropes, illustrated specifically by a decrease in their level of proliferation. This is the first presentation of evidence showing that dopaminergic innervation within the intermediate lobe of the rat pituitary regulates melanotrope proliferation.
Trophic influences of human and rat amniotic fluid on neural tube‐derived rat fetal cells
Authors: Colombo Jorge A. , Napp Mónica , Depaoli Juan R. , Puissant Virginia
Published: April 2003

Normal human (week 17–20) and rat (E16–17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions. Phase‐bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi‐automated procedures. Subcultured astroglia were immuno‐reacted against glial fibrillary acidic protein and fibronectin. Rat and human amniotic fluid allowed survival and growth of neuronal and non‐neuronal cells. Human amniotic fluid samples were trophic in variable degrees. Cerebral cortex subcultured astroglia usually expressed a radial‐like morphotype. Although charcoal‐adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment. Growth of cell processes in neuronal‐ and glial‐like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors. It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons. Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid. Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum. The role of amniotic fluid during the early phases of brain organogenesis is discussed.
Laminar expression of m1‐, m3‐ and m4‐muscarinic cholinergic receptor genes in the developing rat visual cortex using in situ hybridization histochemistry. Effect of monocular visual deprivation
Authors: Roβner Steffen , Kues Wilfried , Witzemann Veit , Schliebs Reinhard
Published: April 2003

The postnatal development of laminar pattern of m1‐, m3‐ and m4‐mRNA‐muscarinic acetylcholine receptor subtypes in the visual cortex of both normally raised and monocularly deprived rats (one eyelid sutured at the age of 11 days) was studied using in situ hybridization histochemistry and computer‐assisted image analysis. From birth until day 15 the level of m1‐receptor transcript in layer II/III increases markedly as compared to deeper layers. From day 15 up to day 18 a transient bimodal pattern develops with peaks in layers II/III and VI. Already on day 35 a more homogeneous distribution of m1‐receptor mRNA level is detectable persisting until adulthood. In contrast, the m3‐receptor mRNA shows already at birth a bimodal distribution with peaks in layers II/III and VI. Further development until adulthood results in transient changes in the ratio of the mRNA levels in these layers. In the adult visual cortex a similar laminar pattern as at birth is observed. From day 1 up to day 10 a relative increase in the mRNA level of the m4‐receptor in layers II to IV is observed. From day 10 until day 15 a bimodal distribution of receptor mRNA develops with peaks in layers III and VI which is similar to the adult stage. However, between days 18 and 35 a shift in the laminar receptor mRNA distribution occurs resulting in peaks in layers IV and VI. The labeling of the m5‐receptor transcript in rat visual cortex was very weak and did not show any alteration with age.

Unilateral eyelid closure from postnatal day 11 resulted in transient changes in the laminar distribution of m3‐ and m4‐receptor mRNA between postnatal days 18 and 25, whereas the development of the laminar pattern of the m1‐receptor mRNA was not affected regardless of the length of visual deprivation.

The distinct laminar developmental pattern of mRNA muscarinic receptor subtypes in rat visual cortex suggests specific roles of the muscarinic receptor subtypes during the first weeks of postnatal maturation of visual function.
Effects of early chronic diazepam treatment on incorporation of glucose and β‐hydroxybutyrate into cerebral amino acids: Relation to undernutrition
Authors: Schroeder Henri , Collignon Alain , Uttscheid Laurent , de Vasconcelos Anne Pereira , Nehlig Astrid
Published: April 2003

The effects of early chronic diazepam (DZP) exposure on blood glucose and ketone body concentration and glucose and β‐hydroxybutyrate (βHB) utilization for regional cerebral amino acid biosynthesis were studied in suckling rats. The animals were treated from postnatal day 2 (P2) to 21 (P21) by a daily subcutaneous injection of 10 mg/kg DZP or of the dissolution vehicle and studied at P5, P10, P14 and P21, together with an additional group of food‐restricted rats obtained by an increase in litter size. DZP treatment induced a 9–26% decrease in body and brain weight. Undernutrition decreased body weight by 20–24% at all ages whereas brain weight was relatively spared. DZP and N‐desmethyldiazepam concentrations decreased with age and were cleared from brain and plasma by 6–8 hrs after the injection. DZP decreased plasma glucose concentrations by 6–12% at P5, P14 and P21, whereas undernutrition did not change plasma glucose concentrations, except for a 7% decrease at P14. DZP treatment had no consequences on circulating concentrations of both ketone bodies while undernutrition increased their concentration by 45–362% at all ages. The conversion of [14C]glucose into cerebral amino acids was reduced by DZP at P5 and P10. The cerebral concentration of neurotransmitter amino acids was not affected by DZP treatment which only increased the amount of neutral amino acids mainly in the cerebellum at P5 and P10. After [U‐14C]glucose injection, specific radioactivities of cerebral amino acids were mostly decreased by DZP from P5 to P14 and significantly increased at P21. With [3‐14C]βHB as a precursor, specific radioactivities of neurotransmitter amino acids were increased by DZP. In conclusion, P5 and P10 rats appear to be most sensitive to DZP effects whereas some tolerance to the drug seems to develop by P21. The lack of effects of DZP on blood ketone body concentrations compared to food restriction as well as the relative sparing of brain weight in undernourished rats confirms that the cerebral metabolic consequences of early DZP exposure on brain energy metabolism are mostly direct effects not mediated by sedation‐induced undernutrition.
Biochemical properties of Na+/K+‐ATPase in axonal growth cone particles isolated from fetal rat brain
Authors: Mercado R. , Hernández‐R. J.
Published: April 2003

Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N+/K+‐ATPase. Kinetic assays of the enzyme in AGC showed that Km values for ATP or K+ are similar to those reported for the adult brain enzyme. For Na+ the affinity (Km) was lower. Vmax for the three substrates was several times lower in AGC as compared to the adult value. We also observed two apparent inhibition constants of Na+/K+‐ATPase by ouabain, one of low affinity, possibly corresponding to the α1 isoform and another of high affinity which is different to that described for the α2 isoform of the enzyme. These results support an important role for the sodium pump in the mantainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na+/K+‐ATPase in AGC is in a transitional state towards the adult configuration.