bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural study of calycine synaptic endings of colossal vestibular fibers in the cristae ampullares of the developing chick
Authors: Peusner Kenna D. , Lindberg Nancy H. , Mansfield Paul F.
Published: April 2003

Bipolar vestibular ganglion cells give rise to the colossal vestibular fibers in the chicken. These fibers form the largest calycine endings in the cristae ampullares and also the spoon endings in the tangential vestibular nucleus of the medulla oblongata. Because these synaptic endings are two of the largest and most distinctive in the vertebrate nervous system, they are especially suitable for comparisons of the development of synapses and synaptic endings of a specific cell type. An ultrastructural study of the spoon endings and quantitative data on their synapses were available from material of 15‐day‐old chick embryos, hatchlings, and 3‐yr‐old chickens. Here we provide similar data on the large calyces. Briefly, large calyces exhibited no ultrastructural changes corresponding to the changes in the spoon endings apparent when they retract from their target cell surfaces around hatching time. However, the concentration of the ribbon synapses at the large calyces decreased around hatching, when the concentration of the chemical synapses at the spoon endings declined. Moreover, the concentration of the ribbon synapses at the large calyces corresponded closely to the concentration of the chemical synapses at the spoor endings at the same age. Thus at the developmental ages studied, there were similar concentrations in the peripheral and central synapses formed at two different synaptic endings, both derived from one cell type and participating in the same neural pathway.

These findings raise the issue of how synapses are regulated locally, but also suggest the possibility for central‐peripheral interactions to produce correlative changes in parallel.
Ontogenetic changes in the level and subcellular distribution of protein kinase C in cat visual cortex
Authors: Stichel C. C.
Published: April 2003

Ontogenetic changes of the total activity and the subcellular distribution of Ca2+‐activated, phospholipid‐dependent protein kinase (protein kinase C) were investigated in the cat visual cortex.

Following homogenization of the tissue in Ca2+‐free buffer and separation of membrane‐bound and soluble protein kinase C, the enzyme was partially purified by diethylaminoethyl‐cellulose chromatography. Total, membrane‐bound and soluble enzyme activity were determined by measuring the phosphate incorporation into lysine‐rich histone, a substrate for protein kinase C.

It was found that the total activity level, while being low in the first 4 weeks, increased rapidly to peak values at 5 weeks of age and declined slightly thereafter. Although at all developmental stages most of the enzyme was recovered in the soluble fraction, the membrane‐bound activity exhibited a considerable increase at 5 weeks of age, reflecting a relative shift of protein kinase C from the cytosol to the membranes.

The increase and redistribution of protein kinase C activity coincide in time with the developmental phase during which the visual cortex is particularly susceptible to undergo use‐dependent modifications. This finding is compatible with the hypothesis that protein kinase C‐mediated phosphorylation processes are involved in activity‐dependent modifications of neuronal transmission.
A morphological and biochemical study of the myelin‐like membrane structures formed in cultures of pure oligodendrocytes
Authors: Nussbaum J. L. , de los Monteros A. Espinosa , Pari F. M. , Doerr‐Schott J. , Roussel G. , Neskovic N. M.
Published: April 2003

This study reports the production of myelin‐like membranes in oligodendrocyte subcultures derived from 20‐day‐old primary glial cell cultures of newborn rat brain. These multi‐layered structures show a variable number of membrane turns; up to 10 concentric lamellae are found in 3‐ to 4‐week‐old subcultures. When they are compacted, alternate dense and intraperiodic lines with a periodicity of 11.2 nm are noticeable. The most typical myelin proteins were detected straight on the multi‐lammellar structures by a gold immunocytochemical method. Subcellular fractions containing these myelin‐like structures were isolated by ultracentrifugation on a discontinuous sucrose gradient. They were analysed by sodium dodecylsulfate‐polyacrylamide gel electrophoresis and immunoblotting; UDP‐galactose: ceramide galactosyltransferase and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase activities were also measured.

The results indicate that the multi‐layered membrane profiles have many characteristics of the myelin found in vivo; nevertheless some differences were still apparent. Our data support the concept of the cultured oligodendrocytes expressing the intrinsic myeltnogenic properties and possessing a basic developmental program of myelination, apparently in the absence of stimuli coming from other brain cells.
Cytodifferentiation and developing neuronal circuitry in the human lateral geniculate nucleus
Authors: Wadhwa Shashi , Bijlani Veena
Published: April 2003

The developing neural substrate in dorsal lateral geniculate nucleus of human fetuses and premature, full‐term and postnatal infants, has been analysed using rapid Golgi impregnation, computerized image analysis, electronmicroscopy, and immunocytochemical method for localization of γ‐aminobutyric acid. Nuclear and cytoplasmic neuronal maturation is observed to extend over the entire period studied. Axodendritic synapses, presumably of retinal origin, are occasionally seen at 13–14 fetal weeks. They become increasingly apparent at 18–19 weeks. Dendrodendritic contacts are visualized at 15 weeks. Cortical terminals and occasional triadic contacts are evident around 21 weeks. The inhibitory interneurons containing γ‐aminobutyric acid are present in small numbers at 15–16 weeks; their numerical density increases considerably at 17 weeks but decreases thereafter. The presence of γ‐aminobutyric acid containing nerve cells and synaptic triads is indicative of the formation of inhibitory circuitry. At 15–16 weeks neurons are mostly bipolar although different forms of multipolar cells may be seen. By 24 weeks the radiated and bitufted multipolar neurons, neurons with beaded dendrites and neurons with axon‐like dendritic processes are identifiable. There are no apparent differences in differentiation of neurons between the cranial and caudal parts of dorsal lateral geniculate nucleus. At 15–16 weeks, however, the cells of prospective magnocellular zone appear to be more mature than do the cells in the parvocellular zone. The neuronal soma increases continuously in size. Dendrite development starts at 15–16 weeks of gestation, thereafter the increase in number of their branches and their length is observed. Between 15–16 and 24 weeks, spines and filiform processes appear first on the proximal shafts of the dendrites and subsequently on their distal portion. There is increase in the number of filiform processes and hair‐like appendages on geniculate neurons of premature infants born at 32 and 37 weeks of gestation and of a 4‐day postnatal infant. Computerized quantitative data substantiate the progressive increase in growth parameters. The significance of comparative and functional aspect of the data is discussed.
Udpgalactose:Ceramide galactosyltransferase in cultured oligodendrocytes: An enzymological and immunological study
Authors: Neskovic Nenad M. , de los Monteros Araceli Espinosa , Roussel Guy , Nussbaum Jean‐Louis
Published: April 2003

The developmental expression of UDPgalactosexeramide galactosyltransferase (CGalT), an enyme marker of one myelinogenic activity in nervous tissue, was studied in cultured oligodendrocytes. The activity of CGalT in cultures followed a characteristic pattern of developmental changes. In the primary cultures these changes could be represented by a biphasic curve with a maximum of enzymatic activity at about the 25th day in culture. After purifying the oligodendrocytes from the primary cultures and replating them in culture dishes, similar developmental changes of CGalT were observed. In the subultures prepared from 20‐day‐old primary cultures the activity of CGalT per oligodendrocyte increased from 1.3 × 10−6 nmol/hr on day 4 to 3.7 × −6nmol/hr on day 21. Immunocytochemical studies with the antiserum against rat brain CGalT showed the presence of CGalT+ oligodendrocytes after 7 days in the primary culture (earliest time studied), later on the number of CGalT+ oligodendrocytes increased until 28 days (latest time examined). In the subcultures of purified oligodendrocytes the bulk of oligodendrocytes was stained by the anti‐CGalT antibodies after 15 days. These results suggest that the initial expression of CGalT in oligodendroglial cultures involves an increase of the number of CGalT+ oligodendrocytes and of the amount of enzyme protein per cell.
Catecholaminergic cells and support cell precursors in neural crest cultures differentially express nerve growth factor receptors
Authors: Bernd Paulette
Published: April 2003

Long‐term neural crest cultures grown in the continuous absence of exogenous nerve growth factor (NGF) contain a subpopulation of cells with NGF receptors exclusively of the low affinity subtype (d of approximately 3.2 nM). The current studies combined immunocytochemistry, using GIN1 (a support cell marker) or tyrosine hydroxylase antibodies, with radioautography following exposure to iodinated nerve growth factor (125I‐NGF). The majority of cells specifically binding 125I‐NGF were found to be immunoreactive for GIN1, indicating that the primary cell phenotype expressing receptors for NGF appear to be support cell precursors, at least under these conditions. These cells are likely to be responsive to and/or dependent upon NGF; the nature of this response or dependency remains to be determined. Some cells exhibiting silver grains were not immunoreactive for GIN 1, suggesting that other cell phenotypes in neural crest cultures also have NGF receptors. In addition, some neural crest cells were found that stained with GIN1 and lacked 125I‐NGF binding. Tyrosine hydroxylase‐like immunoreactive cells apparently did not bind 1251‐NGF under these culture conditions. Catecholaminergic sympathetic and sensory neurons from embryonic ganglia, derived from the neural crest, express both the high and low affinity forms of the NGF receptor. In order to determine whether the microenvironment played a role in the type of Catecholaminergic cells appearing in culture, neural crest cells were grown in the continuous presence of exogenous NGF. Under these conditions, many tyrosine hydroxylase‐like immunoreactive cells were found that specifically bound 125I‐NGF. In addition, silver grains were still detected on these cells following a chase with nonradioactive NGF, designed to eliminate 125I‐NGF bound to low affinity sites. Therefore, the Catecholaminergic cells possess both the low and high affinity forms of the receptor. NGF's ability to modulate tyrosine hydroxylase activity, as it does in mature Catecholaminergic neurons, was tested in this system. Surprisingly, there was no statistically significant difference in tyrosine hydroxylase activity in cultures grown in the absence or presence of exogenous NGF. This raises the possibility that embryonic Catecholaminergic cells are unable to respond to NGF in this specific way, even though the receptors for the factor are present.
Selective effects of thyroid hormonal deprivation on growth and development of olfactory receptor sheet during the early postnatal period: A morphometric and cell count study in the rat
Authors: Paternostro Mark , Meisami Esmail
Published: April 2003

Light microscopic numerical and morphometric studies were conducted on the olfactory epithelium of postnatal normal and hypothyroid rats. The normal rat olfactory epithelium undergoes marked growth and development during the suckling period (days 1–25): thickness, 50%; area, × 8, total number of olfactory neurons, basal and supporting cells, × 10, × 11 and × 8, respectively. The effects of thyroid hormonal deprivation on these proliferative postnatal growth changes were studied by adding PTU (n‐propylthiouracil, a reversible antithyroid goitrogen) to the litter's drinking water from birth to weaning (day 25).

The general architecture of naso‐olfactory cavities as well as the histology and thickness of the olfactory epithelium were unaffected in the hypothyroid pups. However, the surface area of the olfactory receptor sheet was reduced by 40%, the reduction occurring throughout the cavity, though not uniformly. The total number of olfactory neurons, supporting and basal cells were reduced by 33, 45 and 47%, respectively. These results indicate that the postnatal vertical accretion of olfactory neurons occurring across the epithelial thickness is unaffected in the hypothyroid pups, while the horizontal proliferation of neurons accompanying the expansion of the sheet's surface area is markedly reduced. The results suggest differential effects of thyroid hormones on these modes of proliferative growth and imply further that in addition to possible direct effects, the influence of thyroid hormones on developmental growth of the olfactory epithelial sheets may be secondary to effects on the underlying submucosal connective tissue.
The time course of hippocampal cholinergic innervation in the developing hypothyroid rat. A combined histochemical and biochemical study of acetylcholinesterase activity
Authors: Rami Abdelhaq , Rabie Alain , Clos Jean
Published: April 2003

Development of cholinergic innervation in the hippocampal formation of normal and hypothyroid rats was studied by a combined biochemical and histochemical analysis of acetylcholinesterase (AChE) activity in normal and hypothyroid rats. The normal developmental pattern of cholinergic activity suggests an entrance of septal cholinergic fibers from the fimbria to different zones of the hippocampal formation mainly during the first postnatal week. By 10 days of age, the regional distribution of staining was similar to that in the adult. Thereafter, the intensity of staining increased without major changes in the laminar organization. As shown by closely related histochemical and biochemical findings, hypothyroidism led to a delay in the arrival of cholinergic afferences and a possible subsequent cholinergic hyperinnervation of the hippocampal formation in adulthood. These results are discussed taking into account the time course of structural development in the two synaptic compartments, namely the extrinsic septal neurons and intrinsic pyramidal and granule cells, in comparison with the development of thyroid function.
Development of the neuronal system containing neuropeptide Y in the rat hypothalamus
Authors: Kagotani Y. , Hashimoto T. , Tsuruo Y. , Kawano H. , Daikoku S. , Chihara K.
Published: April 2003

In the rat hypothalamus, neuropeptide Y‐containing neurons first appeared on day 14.5 of gestation in the arcuate nucleus and in the dorsolateral hypothalamic area. Until birth neuropeptide Y‐containing cell bodies increased in number in the arcuate, dorsomedial‐lateral and paraventricular nuclei, but disappeared thereafter, but some cells remaining in the arcuate nucleus. In animals treated neonatally with monosodium l‐glutamate to destroy the arcuate nucleus, neuropeptide Y‐immunoreactivity became evident in many cells scattered in the magnocellular paraventricular and dorsomediallateral hypothalamic nuclei on day 16 but not on days 60 and 120. These neuropeptide Y‐immunoreactive neurons which appeared in the paraventricular nucleus were also vasopressin‐positive. Neuropeptide Y fibers, on the contrary, remarkably diminished in number on day 16, particularly in the paraventricular and dorsomedial‐lateral nuclei, and the medial preoptic area, but made a considerable recovery on days 60 and 120. Hence it is probable that, in normal ontogenetic progress, the development of the neuropeptide Y fibers in these areas is inhibitorily affected by that of arcuate neuropeptide Y neurons.
Stimulation of choline acetyltransferase in spinal cord explants by limb mesenchyme
Authors: Muhlach William L. , Pollack Emanuel D.
Published: April 2003

Choline acetyltransferase (ChAT) activity in developing spinal cord explants in vitro is shown to be dependent on the presence of co‐cultured immature limb tissue. Frog tadpole spinal cord explants grown on collagen or polylysine expressed stage‐appropriate levels of ChAT activity only when in the presence of the limb mesenchyme target. Neither skeletal muscle nor polylysine, both of which enhance neurite growth accompanied by increases in cord protein, were capable of maintaining the level of ChAT activity characteristic of these spinal cords in vivo. The results demonstrate that developmentally significant levels of ChAT can be maintained in vitro under appropriate conditions that may act in part through the maintenance of cholinergic motor neurons.
Cholinergic modulation of aged‐like retention deficits in young autoimmune mice
Authors: Forster Michael J. , Lal Harbans
Published: April 2003

Separate age groups of autoimmune NZB/BINJ and non‐autoimmune C57BL/BNNia mice were compared for habituation of locomotor activity and its retention over four separate testing sessions spaced at 24‐hr intervals. A decline in locomotion (distance in cm) or in the time spent in the center zone as a function of sessions was taken to indicate retention for habituation to stimuli within the test apparatus. The time spent in the center zone decreased as a function of sessions in young and mature C57BL/6NNia mice but failed to show reliable between‐session decreases in old (24–26‐months) C57BL/ 6NNia mice. When compared with the old C57BL/6NNia mice, young NZB/BINJ mice showed similar impairments. Habituation of locomotion was present in all age groups of C57BL/6NNia mice, but absent in NZB/BINJ mice regardless of age. The retention impairments of 2–4 month old NZB/BINJ mice were attenuated when i.p. injections of 0.04–0.16 mg physostigmine/kg were given just following each habituation session. The effectiveness of physostigmine was substantially reduced when injections were delayed by 20 min or longer following each habituation session. The time‐dependent reversal of the aged‐like retention deficits by the cholinesterase inhibitor, physostigmine, suggests that cholinergic modulation of memory storage processes may be impaired in NZB/BINJ mice.
Effects of NMDA antagonists on developmental plasticity in kitten visual cortex
Authors: Rauschecker Josef P. , Egert Ulrich , Kossel Albrecht
Published: April 2003

The existence of Hebb synapses in the visual cortex of young kittens has long been postulated. A mechanism for the correlation of activity in simultaneously active pre‐ and postsynaptic neurons could be provided by the properties of the N‐methyl‐d‐aspartate (NMDA) receptor and its associated Ca2+ channel, which opens in a transmitter‐ and voltage‐dependent manner. We have studied the effects on cortical plasticity of blocking NMDA receptors in different ways with competitive and non‐competitive NMDA antagonists.

In our first approach, the non‐competitive NMDA antagonist ketamine, a short‐acting dissociative anaesthetic, was injected systemically after each of a series of brief monocular exposures. This procedure prevented the development of an ocular dominance shift towards the experienced eye in the visual cortex. Other short‐acting anaesthetics, such as xylazine or methohexital, while providing the same depth of anaesthesia, did not have the same effect on ocular dominance plasticity. We conclude, therefore, that ketamine quite specifically interferes with synaptic consolidation in the visual cortex.

In order to establish a role of NMDA receptors for cortical plasticity directly in the visual cortex, we performed another series of experiments: 2‐amino‐5‐phosphono‐valerate (APV), a competitive NMDA antagonist, was infused intracortically by means of implanted osmotic minipumps in kittens, which were monocularly deprived for 1–2 weeks. Within a radius of 4–5 mm, the expected ocular dominance shift was prevented or reduced. In addition, however, physiologically determined cell density and responsiveness to visual stimuli were grossly abnormal around the infusion site, and histological cell density was also reduced. Similar effects were found when MK801 (a non‐competitive NMDA antagonist) was used in the same type of experiment.

The outcome of both experimental approaches makes it very likely that NMDA antagonists somehow interfere with cortical plasticity. Their mode of action, however, remains ambiguous. Although it is quite possible that blocking of the NMDA channel prevents the Hebbian correlative process necessary for synaptic consolidation, more complex effects, such as an interference with a neurotrophic action normally exerted via the NMDA receptor, may have to be taken into account as well.
Treatment of mammalian spinal cord injury with antioxidants
Authors: Naftchi N. Eric
Published: April 2003

After spinal cord injury, two groups of cats were treated with a combination of methylprednisolone sodium succinate (MP, 35 mg/kg) and epsilon‐aminocaproic acid (EACA, 350 mg/kg), and guanabenz acetate (0.65 mg/kg). Guanabenz acetate was administered twice daily for 8 weeks. In the first group, the treatment significantly increased blood flow in the abdominal aorta. All cats treated with guanabenz acetate 3 hr after spinal cord contusion had return of micturition and none suffered complete paraplegia. Four animals had partial and the other four had complete motor recovery. A superoxide (O2−) generating system, horseradish peroxidase, decreased [14C]gamma amino butyric acid uptake by mouse cortical slices by 33% but when superoxide dismutase was added to the medium, the uptake was reduced by only 9%. The nerve endings were also protected by superoxide dismutase from morphologic damage by (O2−) as observed by electron microscopy. The agents used in these studies produce their ameliorating effects by virtue of their anti‐inflammatory, antioxidant. and membrane stabilizing properties, and enhancing the regional microcirculation. In addition to having these properties, guanabenz acetate is also an alpha2 adrenoceptor agonist.
Iron‐induced lipid peroxidation and brain injury responses
Authors: Willmore L. James , Triggs William J.
Published: April 2003

Head trauma with cerebral contusion causes extravasation of red blood cells, followed by hemolysis and deposition of iron‐containing blood products within the neuropil. Liberation of heme compounds is associated with deposition of hemosiderin, and with gliosis, neuronal loss and occasionally the development of seizures. In this experiment we injected components of red blood cell contents into rat amygdala, and then measured the rate of appearance of products of lipid peroxidation. Injection of microliter volumes of hemin and hemoglobin, with hematoprotoporphyrin and rodent plasma injection and contralateral uninjected tissue as controls, showed that the presence of the iron moiety within the protoporphyrin ring was required to initiate and propagate peroxidation. Free radical reactions initiated by iron or heme deposited within the neuropil may be a fundamental reaction associated with brain injury responses, and possibly with posttraumatic epileptogenesis.
Free radical scavenging systems in developing rat brain
Authors: Shivakumar Bangalore R. , Anandatheerthavarada Hindupur K. , Ravindranath Vijayalakshmi
Published: April 2003

Because the developing brain is subject to high oxygen tension and lacks a functional blood‐brain barrier, anti‐oxidant protection is important to development in the brain. The levels of superoxide dismutase, copper‐zinc superoxide dismutase. manganese superoxide dismutase. catalase, glutathione and related enzymes, namely, glutathione reductase and glutathione peroxidase were determined in rat brain at various stages of development. The levels of thiobarbituric acid reactive products, indicative of lipid peroxidation, were very low at birth and increased to adult levels by the 16th day after birth. Brain glutathione levels displayed significant variations during the first 2 weeks after birth but not thereafter. Catalase activity in developing brain slowly increased over 45 days. Total superoxide dismutase activity in 1‐day‐old rat brain, 80% of the adult rat brain level, subsequently decreased on day 6. Total superoxide dismutase activity, however, increased again in 10‐day‐old rats and remained constant thereafter. While the developmental pattern of manganese superoxide dismutase was similar to that of the total superoxide dismutase, the copper‐zinc superoxide dismutase levels were low at birth and reached adult levels on the 10th day after birth. There was no variation in glutathione reductase and peroxidase levels except for a decrease on day 16 of glutathione reductase and slow increase in adult levels by day 28. The present findings suggest that the overall levels of antioxidant enzymes in the developing brain are comparable to a large extent to those present in the adult brain. In contrast to the developing brain, hepatic levels of glutathione, total superoxide dismutase, manganese superoxide dismutase are significantly lower at birth and increase during development.
Transient appearance of beta‐endorphin immunoreactive cells within the germinal zone of neonatal rat forebrain
Authors: Loughlin Sandra E. , Kornblum Harley I. , Massamiri Tania , Leslie Frances M.
Published: April 2003

Recent evidence implicates endogenous opioid systems in basic processes which underlie morphogenesis. The present report describes a population of cells within the germinal zone of the neonatal rat forebrain which are immunoreactive for the opioid peptide β‐endorphin and other peptides derived from the proopiomelanocortin precursor. These cells are present at the time of birth, but are no longer detectable by the sixth postnatal day. They have medially and laterally directed processes which extend to the ventricular wall and across the caudate putamen to its lateral border. Cells of similar morphology and distribution which are immunoreactive for two other proopiomelanocortin peptides, α‐melanocyte stimulating hormone and adrenocorticotrophic hormone, were also observed in similar distributions during the same developmental period. These data are consistent with the hypothesis that cells within the germinal zone transiently synthesize proopiomelanocortin, which is further processed to yield these three peptide products. This finding may be important in understanding the role of proopiomelanocortin‐derived peptides in neural development.
Growth factors attenuate the cholinotoxic effects of ethanol during early neuroembryogenesis in the chick embryo
Authors: Brodie Chaya , Kentroti Susan , Vernadakis Antonia
Published: April 2003

The interaction between growth factors and ethanol on cholinergic neuronal expression was studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase activity as a cholinergic marker. As we have previously reported (Brodie & Vernadakis, Dev. Brain Res.56: 223–228, 1990; Kentroti and Vernadakis, Dev. Brain Res.56: 205–210, 1990), ethanol administration in ovo at embryonic days 1–3 produced a 30% decrease in choline acetyltransferase activity. Nerve growth factor and epidermal growth factor administration alone, at embryonic days 1–3, produced a slight increase in choline acetyltransferase activity of both brain and spinal cord when examined at embryonic day 8. Concomitant administration of either nerve growth factor or epidermal growth factor with ethanol eliminated the decrease in choline acetyltransferase activity produced by ethanol. Moreover, administration of either nerve growth factor or epidermal growth factor at embryonic days 4–7 to embryos pretreated with ethanol at days 1–3 raised choline acetyltransferase activity to a level similar to that observed in controls. Thus the growth factors reversed the ethanol‐induced cholinergic insult and restored the cholinergic population to normal.

These findings provide evidence of a possible role of NGF and EGF in interfering with the neurotoxic effects of ethanol during embryogenesis.
Developmental changes in B‐50 (GAP‐43) in primary cultures of cerebral cortex: B‐50 immunolocalization, axonal elongation rate and growth cone morphology
Authors: Ramakers G.J.A. , Oestreicher A.B. , Wolters P.S. , van Leeuwen F.W. , De Graan P.N.E. , Gispen W.H.
Published: April 2003

Changes in neurite outgrowth parameters and in the immunolocalization of the neuronal growth‐associated protein B‐50 (GAP‐43) were studied in cultured neocortex as a function of development. In addition, we studied the effects of chronic blockade of bioelectric activity (BEA) with tetrodotoxin (TTX) on these parameters.

Axonal outgrowth rate in control cultures reached a maximum at 8 days in vitro (DIV) and declined to a low level at 21 DIV. B‐50 staining shifted from the perikaryon to the axons and growth cones during the first 3 DIV. In axons the intensity of B‐50 staining increased towards the growth cone. Within growth cones, the central/basal region and filopodia were intensely stained, whereas lamellipodia showed only marginal staining. Growth cone size gradually decreased after 3 DIV, due to the successive loss of lamellipodia and filopodia, and became club‐shaped during the second week, until by 21 DIV growth cones were completely lost, and axons started retracting and degenerated. In the central area of the cultures, growth cones also decreased in size with time, but became stabilized as presynaptic elements onto other neurons.

Acute addition of TTX did not affect the outgrowth rate at 6 DIV. Chronic TTX treatment led to an earlier retraction and degeneration of axons than in control cultures and to a loss of B‐50‐stained cells and varicosities during the third week, but did not affect growth cone morphology or B‐50 staining. The regressive phenomena are probably due to an increased neuronal cell death shown to occur after chronic TTX treatment. The developmental changes in axonal elongation rate and growth cone morphology may be related to developmental changes in the content and/or phosphorylation of B‐50 (GAP‐43, which are studied in the same cultures in the following paper [Ramakers et al. (1991) Int. J. Devl Neurosci.9, 231–241].
Activity of hippocampal extract on development of [3H]5‐HT high‐affinity uptake in dissociated microcultures
Authors: Azmitia E.C. , Lama P. , Segal M. , Whitaker‐Azmitia P.M. , Murphy R.B. , Zhou F.C.
Published: April 2003

Specific and localized lesions of the 5‐HT fibers in the hippocampus induce homotypic collateral sprouting and enhance serotonergic fiber outgrowth from adult neurons and transplanted fetal tissue. In this study, hippocampal extracts were prepared and applied to primary cultures of fetal serotonergic neurons. The effects of plating density and serum additives were examined. The growth of the serotonergic neurons in the rostral brainstem dissociated cultures were estimated by measuring the specific uptake of [3H]5‐HT. The results indicate the presence of a trypsin‐sensitive factor which is active when prepared fresh at dilutions up to 1/10,000. The factor is higher in hippocampus than cerebellum. Young male tissue contained more activity than either female or aged hippocampus. Although both positive and negative effects are described, higher dilutions of factor (1/1,000) were generally stimulatory in high density cultures while lower dilutions (1/10) were inhibitory in low density cultures. Specific removal of 5‐HT hippocampal afferents with fornix‐fimbria microinjections of 5,7‐dihydroxytryptamine resulted in an initial loss of activity (2 days and 2 weeks) followed by an enhanced activity (2 months) compared to normal hippocampal extract. Several possibilities are discussed as to the identity of the serotonergic growth factor from hippocampal supernatant.
DIfferential effects of prenatal and postnatal acth or nicotine exposure on 5‐HT high affinity uptake in the neonatal rat brain
Authors: King Jean A. , Davila‐Garcia Martha , Azmitia Efrain C. , Strand Fleur L.
Published: April 2003

These studies were designed to examine the differential effects of prenatal or postnatal administration of ACTH 1‐39 and nicotine, on 5‐HT high affinity uptake in brainstem and hippocampal synaptosomes. ACTH was administered prenatally (to pregnant dams) and postnatally to the neonates. Postnatal administration of ACTH significantly increased high‐affinity 5‐HT uptake in the hippocampus and especially the brainstem at both 7 and 21 days after birth. Prenatal ACTH, on the other hand, transiently increased 5‐HT uptake in only the brainstem at 7 days, a change that was reversed at 21 days. While the effects of postnatal nicotine administration were essentially the same as those of postnatal ACTH treatment, prenatal nicotine, unlike ACTH, did not alter 5‐HT uptake in 7‐day‐old rats but did reduce uptake in both tissues at 21 days. The observation that postnatal nicotine mimics the effects of postnatal ACTH and that nicotine stimulates ACTH release, suggests that the postnatal effects of nicotine may be exerted through ACTH.
Fetal alcohol delays the developmental expression of myelin basic protein and transferrin in rat primary oligodendrocyte cultures
Authors: Chiappelli F. , Taylor A.N. , de los Monteros A. Espinosa , de Vellis J.
Published: April 2003

This study has examined the development of immunoreactive myelin basic protein and transferrin in primary glial cell cultures. Cultures were initiated from control and experimental Sprague‐Dawley rats 1–2 days postnatally. Experimental treatment involved exposure to 5% (w/v) ethanol in a liquid diet during the last two weeks of gestation. Prenatal alcohol administration delayed the expression of myelin basic protein and transferrin during the first three weeks postnatally. Other oligodendroglial and astroglial markers were little affected, if at all, by fetal alcohol exposure.
The structural and neurochemical development of the fetal guinea pig retina and optic nerve in experimental growth retardation
Authors: Rees Sandra , Bainbridge Andrew
Published: April 2003

In this study we have examined structural and neurochemical aspects of retinal and optic nerve development in experimentally growth‐retarded fetal guinea pigs following maternal unilateral artery ligation. Eye weight (n = 4) and total retinal area (n = 6) at 62 days gestation (term ∼66 days) were both relatively spared when expressed as a percentage of body weight but in absolute terms were significantly reduced by 18% (P<0.001) and 13% (P<0.05) respectively when compared with age‐matched controls.

The numerical density of neurons in the ganglion cell layer was significantly higher at both 52 days (n = 4) and 62 days (n = 4) in growth‐retarded fetuses compared with controls. However, there was no difference between the groups in the total number of neurons in this retinal layer at either age, since retinal areas are reduced in growth retardation. The area of neuronal somata in the ganglion and inner nuclear layers was significantly reduced in growth‐retarded fetuses compared with controls. There was a concomitant reduction in the width of the cellular layers in the retina and also in the plexiform (synaptic) and photoreceptor layers. The growth of the outer segments of the photoreceptor layer was particularly affected in peripheral retina. The higher packing density of cells and the reduced growth of the plexiform layers suggests a reduction in the growth of the neuropile in growth‐retarded fetuses compared with controls. The radial bundling of ganglion cell axons coursing across the retina to enter the optic nerve head was poorly defined in growth retardation. In addition myelination was delayed in the optic nerve with the numerical density of myelinated axons being significantly reduced (P<0.005) in growth‐retarded fetuses compared with controls.

There was a significant reduction (P < 0.01) in the number of amacrine cells in the inner plexiform layer expressing Substance P‐like immunoreactivity in growth‐retarded fetuses compared with controls. Glutamate‐like immunoreactivity was most intense in the five laminae of the inner plexiform layer and in the outer plexiform layer and less pronounced in photoreceptors, ganglion cells and their axons. There was no qualitative difference in glutamate immunoreactivity between control and growth‐retarded fetuses in any of these structures.

Thus we have shown that intrauterine growth retardation has specific effects on the development of the fetal guinea pig retina, reducing the growth of several types of neurons and their processes and affecting the expression of the neuropeptide substance‐P in amacrine cells.
NMDA receptor levels in chronically depolarized long‐term neonatal rat neocortical explants
Authors: Baker R.E. , Vermeulen J.P. , Boer G.J.
Published: April 2003

The levels of the N‐methyl‐d‐aspartate subclass of glutamate receptor were determined in organotypic neocortical explants chronically exposed to a growth medium containing 25 mM potassium (K25). Explants exposed to 25 mM potassium for 2–3 weeks evinced significantly less binding of the non‐competitive N‐methyl‐d‐aspartate receptor‐associated channel antagonist 125I‐MK801 than did age‐matched controls. Surprisingly, cultures that were returned to control growth medium for a further 2 or 7 days showed even less binding of the ligand. The Kd values of binding were not affected and were similar to those of fresh postnatal cortex. The maximum number of binding sites did not vary between postnatal day 6 and 14 days in vitro control cultures, but were significantly less than those measured at postnatal day 20 (comparable age: 14 days in vitro).

Several conclusions can be drawn from these findings: (i) the density of the N‐methyl‐d‐aspartate sub‐class glutamate receptor does not attain in vivo levels under the present culturing conditions, but remains at those levels associated with the stage of development at which the tissue was brought into culture, (ii) chronic depolarization results in a drastic reduction in N‐methyl‐d‐aspartate receptor density, which is not compensated for after the return to normal growth conditions, (iii) depolarization selectively inhibits cellular maturation of the neocortex (but not survival, as shown previously), including neurotransmitter receptor production and/or insertion into membranes or assembly.
Development of phenylethanolamine N‐methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata
Authors: Bohn Martha Churchill , Engele Jürgen
Published: April 2003

The adrenergic phenotypic marker, phenylethanolamine N‐methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons.

Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT‐immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT‐IR neurons were observed in cultures prepared only from embryos of 46–51 somites (embryonic day E13–13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16. In cultures of the E13 rat, PNMT was found to be catalytically active at 4 days in vitro and the levels of PNMT activity per neuron, as estimated from the number of PNMT‐IR neurons in sister cultures, increased 2‐fold in cultures grown for 8 days in the presence of 10% fetal bovine serum and 1.5‐fold in defined, serum‐free conditions. Treatment of cultures with either corticosterone or dexamethasone had no effect on PNMT activity, supporting previous studies suggesting that PNMT in central neurons is not regulated by glucocorticoids.

These studies provide a convenient and defined in vitro system for studying factors that influence the development of central adrenergic neurons. The differential development of PNMT‐IR and TH‐IR neurons in these cultures suggests that the factors involved in adrenergic neuron survival and/or differentiation are not common to catecholaminergic neurons in general.
Neuronal sprouting and synapse formation in response to injury in the mouse organ of corti in culture
Authors: Sobkowicz Hanna M. , Slapnick Susan M.
Published: April 2003

The effect of mechanical injury on induction of regenerative phenomena within the neurosensory epithelium was investigated in cultures of neonatal mouse cochlea. The oldest examined culture in which new neuronal growth followed insult, was injured at 13 days in vitro and fixed 24 h later. By far, the most vigorous regenerative reaction was observed in a 3‐day culture 4 h post‐injury. The reaction included sprouting of nerve fibers injured directly, synapse formation between the surviving hair cells and sprouting neuronal growth cones, wrapping of growing nerve fibers by extending processes of hair cell cytoplasm, and collateral sprouting of synaptically‐engaged nerve endings and of nerve fibers in passage.
Effects of acetyl‐l‐carnitine on the survival of adult rat sensory neurons in primary cultures
Authors: Formenti A. , Arrigoni E. , Sansone V. , Martelli E. Arrigoni , Mancia M.
Published: April 2003

Acetyl‐l‐carnitine produces a significant increase in the survival time‐course of adult rat sensory neurons maintained in primary cultures up to 40 days. The analysis of our data suggests that 200 μm acetyl‐l‐carnitine added to the medium, slows down neuronal decay especially in the first 10 days in vitro, sparing a fraction of cells which would otherwise be lost.

Patch‐clamp recordings from these neurons show that superfusion with acetyl‐l‐carnitine (100–1000 μM) does not induce any membrane current. In addition an agonist muscarinic effect particularly concerning high‐voltage activated calcium channel modulation appears to be ruled out.

In conclusion our data favour the role of acetyl‐l‐carnitine in the trophism of sensory neurons in adult rats. In agreement with other in vivo experiments our data reinforce the hypothesis that this substance might be involved in reducing neuronal loss observed in nervous system aging.
Hippocampal cholinergic alterations and related behavioral deficits after early exposure to ethanol
Authors: Pick Chaim G. , Cooperman Michael , Trombka David , Rogel‐Fuchs Yael , Yanai Joseph
Published: April 2003

The present study was designed to ascertain septohippocampal cholinergic alterations and their related behavioral deficits after early exposure to ethanol. Mouse pups were exposed to ethanol, 3 g/kg by daily subcutaneous injection on postnatal days 2–14. At age 50 days, the ethanol‐exposed mice had significant reductions from control levels in eight‐arm maze performance. For example, on the fourth testing day, the number of correct entries in the ethanol group was 21% below control levels (P < 0.05) and the number of trials needed to enter all arms was 48% above control (P < 0.001). It took the ethanol‐exposed mice twice the time to reach criterion than it did control (P < 0.01). A 33% increase from control level in muscarinic receptor number (βmax) was found in the treated mice of age 22 days and a 64% increase at age 50 days (P < 0.001). However, no differences between control and treated groups could be detected in the presynaptic component of the cholinergic innervation, choline acetyltransferase activity. The results suggest that early ethanol exposure acts on hippocampal function similarly to phenobarbital, probably via alterations in postsynaptic processes in the septohippocampal cholinergic pathways.
Culture of rat brain preoptic area neurons: Effects of sex steroids
Authors: Takagi Keiichi , Kawashima Seiichiro
Published: April 2003

The aim of the present study was to investigate the direct effects of testosterone (T), estradiol‐17α (E2) and 5α‐dihydrotestosterone (5α‐DHT) on the survival of neurons and the morphology of neuronal processes (the total process length, the number of processes and the number of process branchings) of the cells derived from neonatal rat preoptic area (POA) to analyze the bases of morphological sexual dimorphism of the POA. The POA cells were exposed to sex steroids during 2–14 days of culture in serum‐free medium. Neuronal survival was significantly greater in cultures treated with 1.0 nM T than controls. Although T at any concentration tested (0.1,1.0 or 10.0 nM) failed to affect the mean value of the process length, a significant difference was observed in the frequency distribution of total process length between 1.0 nM T‐treated cultures and control cultures. By 1.0 nM T, the number of process branchings was also significantly increased. In contrast, the number of neurons and the total process length of neurons were not affected by 5α‐DHT. However, the number of process branchings was significantly increased by 0.1 nM 5α‐DHT. E2 had no significant effect on any of these parameters. These results suggest that T directly enhances the survival of neurons and increases the dendritic and/or axonic field of the POA, which serves, at least partly, for the development of morphological sexual dimorphism in the rat.
Functional PAF receptors in glia cells: Binding parameters and regulation of expression
Authors: Brodie Chaya
Published: April 2003

Platelet activating factor is a unique phosphoglycerine which possesses a variety of biological functions exerting its biological effects via specific surface receptors. In the central nervous system, platelet activating factor has been suggested to play a role during injury especially in conditions of ischemia and trauma‐induced neuronal damage. The specific cell populations expressing platelet activating factor receptor, however, have not been identified. In this study, the binding properties of platelet activating factor receptors in C6 glioma cells and primary cultures of astrocytes and oligodendrocytes were characterized by using the ligand [3H]WEB 2086. Early‐passage glial cells which exhibit oligodendrocytic phenotype, expressed lower levels of [3H]WEB 2086 binding than either late‐passage cells which exhibit astrocytic phenotypes or primary astroglia cells. No specific binding was observed in primary cultures of oligodendrocytes. The Bmax (136 ± 15.3 fmol/mg protein) and Kd (29 ± 3.2 nM) levels obtained for primary astroglia cells were similar to those described for other cell types. The expression of platelet activating factor receptor in early‐passage glia cells was up‐regulated by treatment with insulin which induces astrocytic differentiation. In contrast, db‐cyclic AMP exerted an inhibitory effect on the level of platelet activating factor receptor in both early‐ and late‐passage cells. The level of functional platelet activating factor receptor in C6 cells as measured by the ability of platelet activating factor to induce 45Ca2+ influx was increased in cells expressing astrocytic phenotypes and was decreased in db‐cyclic AMP‐treated cells. In accordance with lack of specific [3H]WEB 2086 binding, platelet activating factor did not induce a detectable response of Ca2+ influx in cultures of oligodendrocytes. This report provides the first direct demonstration of selective expression of functional platelet activating factor receptors and their properties in astroglia cells. The findings support the suggestion that platelet activating factor may play an important role as a mediator of injury and immune responses in the nervous system.