bluuebunny/crossref_metadata_2025 huggingface dataset:
Pre‐ and postnatal development of the small intensely fluorescent cells in the rat superior cervical ganglion
Authors: Soinila Seppo
Published: April 2003

A fluorescence microscopical study has been performed in the pre‐ and postnatal development of the sympathetic superior cervical ganglion of Sprague‐Dawley rat. Ganglia from 10.5‐ to 21.5‐day‐old embryos and newborn to 90‐day‐old postnatal rats were freeze‐dried and the catecholamine‐containing cells were demonstrated by formaldehyde‐induced fluorescence.

The first catecholamine‐containing cells appeared on day 11.5 of gestation. The ganglia of 11.5‐ to 12.5‐day‐old embryos contained a continuous range of cells showing weak to bright fluorescence intensities. In the ganglia of 13.5‐ to 14.5‐day‐old embryos few solitary cells or small groups of cells showing bright fluorescence were discernible among weakly fluorescent developing principal nerve cells. The cell numbers of both types markedly increased during the prenatal period while the mean diameter of the brightly fluorescent cells significantly decreased. In the late prenatal ganglia clusters were observed in which a large brightly fluorescent cell was surrounded by other intensely fluorescent cells of various sizes.

There were about 300 small intensely fluorescent (SIF) cells in the ganglia of newborn rats. This number decreased by about 50% during postnatal week 1 and again increased during postnatal week 4 reaching the adult value of about 500 cells/ganglion. During the postnatal development the SIF cells formed clusters, the largest of which contained several hundred cells.

Occurrence of large intensely fluorescent cells during a limited period suggests that this cell type represents a transitional form. It is possible that in the rat the primitive sympathetic cells continue their development along two lines: some cells remain weakly fluorescent and give rise to the principal nerve cells, and others accumulate catecholamine(s) and are transformed, possibly after mitotic divisions, into smaller brightly fluorescent cells which mature into SIF cells late postnatally.
Thyroid hormone dependency of the developing dorsal raphe nucleus but not the superior cervical ganglion. Evidence from intraocular grafting experiments
Authors: Granholm A.‐CH. , Siegel R.A. , Seiger Å.
Published: April 2003

Immature superior cervical ganglia and brain stem pieces containing 5‐hydroxytryptamine neurons of the dorsal raphe nucleus were grafted to the anterior eye chamber of intact and thyroidectomized recipients. The grafts were left in the eye to mature for 4–6 weeks and were measured through the translucent cornea repeatedly to follow survival and growth. Monoaminergic nerve fibre outgrowth from the grafts onto the host irides was evaluated with Falck‐Hillarp monoamine histochemistry on iris whole mounts.

Body weight and plasma concentrations of thyroid‐stimulating hormone monitored the thyroid state of both experimental and control animals. Intraocular survival and growth of immature superior cervical ganglia was not influenced by thyroid hormone deficiency, whereas the size of dorsal raphe nucleus grafts significantly increased during similar conditions. This increased size was entirely counteracted by daily substitution with l‐thyroxine (100 μg/kg, s.c.) to the thyroid deficient host animals. Fluorescence microscopy, including semiquantitative estimations of fibre outgrowth area and axon bundle formation in the irides, revealed no effects of the thyroid hormone deficiency on the peripheral superior cervical ganglia. The outgrowth of 5‐hydroxytryptamine‐containing nerve fibres from the brain stem grafts, on the other hand, showed a 50% decrease in both axon bundle formation and fibre outgrowth area in thyroidectomized hosts. This effect of the thyroidectomy could be completely reversed by daily substitution with l‐thyroxine. These data strongly support earlier reports describing a thyroid hormone dependency for central noradrenaline‐ and dopamine‐containing neurons during similar conditions. Furthermore, the results indicate a role of thyroid hormones for nerve fibre growth during development of intraocularly isolated central but not peripheral monoaminergic neurons.
Astrocytes grown in oculo: Expression of cell morphologies on the iris as revealed by GFA immunohistochemistry
Authors: Björklund H.
Published: April 2003

Using two experimental approaches, the morphology of central astrocytes growing in vivo with the iris as a substratum were studied. When irides with mature intraocular grafts of cortex cerebri or locus coeruleus were stretch‐prepared as whole mounts and processed for immunohistochemistry with antiserum against glial fibrillary acidic protein (GFA), a restricted halo of fluorescent cells and fibers was seen surrounding the grafts. Similarly, injection into the anterior eye chamber of adult rats, of a cell suspension prepared from cortex cerebri of 10‐day‐old rat pups gave rise to both multiple GFA‐positive astrocytic islets of different sizes and cell densities as well as scattered individual cells on the anterior surface of the host iris. In contrast, astrocytes from similar cell suspensions prepared from young adult animals survived very poorly. In both types of experiments, a large variation in cell morphology ranging from immature epitheloid, via large flat cells with few thick processes, to typical mature star‐shaped astrocytes was observed. This morphological variation is in agreement with that reported for similar cells in tissue culture. Immature‐looking cells always had a strong perinuclear fluorescence; an inverse correlation was observed between cell body size and development of cell processes. Likewise, the fluorescence intensity was higher in well‐developed cells as compared to more immature ones.

The morphology of individual cells did not seem to be dependent upon the time in oculo, since no difference was observed between GFA‐positive cells on irides examined 10 days and 6 weeks after injection of a cell suspension. Similarly, a high number of immature‐looking cells was seen in irides with locus coeruleus transplants grafted more than 6 months earlier. Instead, the cell density seemed to be the crucial factor. Thus, star‐shaped, well‐developed cells were seen growing singly or in less dense groups whereas denser areas contained mainly immature‐looking cells.

Astrocytic processes had a tendency to follow the course of already existing intrinsic GFA‐positive bundles and fibers in the iris. However, exceptions to this were occasionally seen. The fluorescence intensity was clearly higher in astrocytes growing on the iris than in intrinsic GFA‐positive structures, suggesting molecular differences between central and peripheral GFA‐immunoreactive material.
Effects of sex steroids on the growth of neuronal processes in neonatal rat hypothalamus‐preoptic area and cerebral cortex in primary culture
Authors: Uchibori Masayuki , Kawashima Seiichiro
Published: April 2003

Dissociated cells of neonatal rat hypothalamus‐preoptic area and cerebral cortex were grown in primary culture, and the effects of sex steroids on neurons were morphometrically studied. In cultures of the hypothalamus‐preoptic area estradiol‐17β or testosterone propionate significantly increased the total neuronal process length as compared with control cultures given no steroids in the medium. Significant stimulatory effects were detected on 1, 2 and 3 days after plating, while, 5a‐dihydrotestosterone failed to show any stimulatory influence. In contrast, none of these steroids was effective in cultures of cerebral cortical cells. Based on the number of processes arising from the soma, neurons were divided into unipolar, bipolar and multipolar neurons. Percentage of multipolar neurons of the hypothalamus‐preoptic area was more in cultures treated with estradiol‐17β or testosterone propionate than the controls. Significant effect was not noted in cultures with 5α‐dihydrotestosterone, while, in cultures of the cerebral cortical cells, effects on sprouting of these steroids were far less marked than in cultures of the hypothalamus‐preoptic area. The present findings substantiate the importance of aromatization of androgen to exert its influence on neuronal process growth in cell culture.
Effects of ischemia on free fatty acids and diacylglycerols in developing rat brain
Authors: Tang Wilson , Sun Grace Y.
Published: April 2003

Post‐decapitative ischemic treatment imposed on the developing rat brain elicited a marked increase in its susceptibility to free fatty acid (FFA) release between 14 and 17 days, an observation similar to that reported by Bazan (Acta Physiol. Lat. Am.21, 15, 1971). Although the level of diacylglycerols (DG) also increased during this period, the extent of the increase was not as obvious as the FFA. Ischemic treatment to rats after 17 days of age elicited increases in DG and FFA enriched in stearic and arachidonic acids. The delayed response in susceptibility of brain tissue to ischemia‐induced changes seems to suggest that the biochemical mechanism(s) responsible for the FFA and DG release is better correlated to events commensurating with synaptogenesis than with myelination.
Perinatal development of adrenergic, cholinergic and non‐adrenergic, non‐cholinergic nerves and sif cells in the rabbit urinary bladder
Authors: Crowe R. , Burnstock G.
Published: April 2003

The development of adrenergic (indicated by catecholamine fluorescence), acetylcholine‐sterase‐positive (possibly cholinergic), non‐adrenergic, non‐cholinergic (indicated by quinacrine fluorescence) nerves and small intensely fluorescent (SIF) cells in the rabbit urinary bladder was examined in foetal (from 23 days of gestation), newborn and adult animals. Acetylcholinesterase‐positive nerve fibres and ganglion cells and quinacrine‐positive ganglion cells were both present on day 23 of gestation, while quinacrine‐positive varicose nerve fibres were first seen on day 24. At foetal age 26 days, 25–38 ganglia containing quinacrine‐positive cells were seen in whole‐mount preparations of detrusor muscle of the bladder. Each ganglion contained 30–40 quinacrine‐positive cells (diameter 20–40 μm). In contrast, only 5–12 ganglia contained acetylcholinesterase‐positive nerve cell bodies at the same foetal age with only 3–20 cells in each ganglion; these figures remained at about the same level from foetal age 23 days to maturity. No catecholamine‐containing nerve cell bodies were seen at any foetal age or in the adult. Adrenergic nerve fibres were not detected until day 28 of gestation, although small intensely fluorescent cells were first observed on day 26 of gestation. In the adult bladder there was a reduction of approximately 25–35% in the number of quinacrine‐positive nerve cell bodies within the ganglia when compared with the ganglia in 1‐day‐old bladders and an increase in nerve fibre density of about 50% when compared with bladders of earlier ages. A reduction of approximately 90% in small intensely fluorescent cells and a 2‐fold increase of adrenergic nerves was also characteristic of the adult bladder, although no changes were observed in the density of the acetylcholinesterase‐positive cell bodies and nerve fibres. It is concluded that catecholamine‐containing, acetylcholinesterase‐positive and non‐adrenergic, non‐cholinergic nerves follow very different developmental patterns in the bladder.
Neurogenesis of the magnocellular basal telencephalic nuclei in the rat
Authors: Bayer Shirley A.
Published: April 2003

Neurogenesis in the magnocellular basal telencephalic nuclei of the rat was examined with [3H]thymidine autoradiography. The experimental animals were the offspring of pregnant females given two injections of [3H]thymidine on consecutive embryonic (E) days (E12–E13, E13–E14, … E21–E22). On postnatal day (P) 60, the percentage of labeled cells and the proportion of cells originating during 24 h periods were quantified at several anatomical levels throughout the magnocellular basal telencephalic nuclei. The neurons of the horizontal limb of the diagonal band originate mainly between E13 and E16 in a combined rostral‐to‐caudal and lateral‐to‐medial gradient. The neurogenetic gradients in the horizontal limb are continued by generation patterns of cells in the vertical limb of the diagonal band‐medial septal complex, the large cells in the polymorph layer of the olfactory tubercle, and the large cells of the anterior amygdaloid area. The substantia innominata originates between E13 and E17 in combined caudal‐to‐rostral and lateral‐to‐medial gradients. The globus pallidus originates between E13 and E17 in combined caudal‐to‐rostral, ventral‐to‐dorsal and medial‐to‐lateral gradients. The entopeduncular nucleus originates between E12 and E14 in a ‘sandwich’ gradient where neurons in the core of the nucleus are older than those in either the anterior or posterior ends. There is an overall superficial (ventral) to deep (dorsal) neurogenetic gradient between the magnocellular basal nuclei present at any given rostrocaudal level. An important finding is that neurogenetic gradients in the individual components of the magnocellular basal nuclei are alike (with the possible exception of the entopeduncular nucleus) indicating they are part of a single system. Finally, evidence is presented that neurogenetic gradients in the magnocellular basal telencephalic neurons can be correlated with their anatomical projections to the cerebral cortex.
Prenatal treatment with 6‐hydroxydopa and DSP 4: Biochemical, endocrinological and behavioural effects
Authors: König Norbert , Serrano Jean‐Jacques , Jonsson Gösta , Malayal Francis , Szafarczyk Alain
Published: April 2003

Biochemical, hormonal, and behavioural aspects of the effects of prenatal treatment (foetal days 18 and 19) with 6‐hydroxydopa (2 × 40 mg/kg) and DSP 4 (2 × 20 mg/kg) have been studied in the rat. The results of the catecholamine assays suggest that the 6‐hydroxydopa treatment produced a transient decrease of noradrenaline concentration in the cerebral cortex, and a long‐lasting decrease in the cerebellum, whereas DSP 4 produced a long‐lasting decrease in the cerebral cortex and a tendency towards increase in the cerebellum. Assays of adrenocorticotropic hormone and corticosterone indicated a general tendency towards increase in base line as well as in moderate stress situations. The results of behavioural tests indicated hyperactivity and/or hyperreactivity, but not restlessness, and partly supranormal sensorimotor performances.
The microtubular apparatus of cerebellar purkinje cell dendrites during postnatal development of the rat: The density and cold‐stability of microtubules increase with age and are sensitive to thyroid hormone deficiency
Authors: Faivre C. , Legrand Ch. , Rabié A.
Published: April 2003

A quantitative ultrastructural study of microtubules in Purkinje cell dendrites of normal and hypothyroid developing rats was performed after fixation either at room or at low temperature (4°C). In normal animals, the density of microtubules and their fold‐stability increased with age, more especially during the period of intense dendritic growth. Thyroid deficiency delayed the appearance of microtubules and still more the acquisition of their fold‐stability. These effects might explain the defects in Purkinje cell dendritic growth and branching observed in hypothyroid animals.
Development of trochlear motor neurons, superior oblique muscle, and neuromuscular junctions following prevention of cell death by myasthenia gravis immunoglobulin
Authors: Sohal G.S. , Swift T.R.
Published: April 2003

Paralysis of embryo during the period of naturally occurring motor neuron death produces an increase in the number of surviving neurons while the progressive differentiation and maturation of target muscle is severely retarded. Application of immunoglobulin G from patients with acquired myasthenia gravis to duck embryos during the period of trochlear motor neuron death also prevents this neuron loss but without paralyzing the embryo. The present study was conducted to investigate whether or not the differentiation and maturation of motor neurons, target muscle, and neuromuscular junctions were retarded following prevention of cell loss with myasthenic immunoglobulin. Immunoglobulin concentrates from myasthenics and normal human volunteers were applied daily to the chorioallantoic membrane of duck embryos from day 10 onward. The development of the trochlear neurons and the superior oblique muscle was examined with light and electron microscopy on embryonic days 12, 16 and 20. The motor neurons at the light and electron microscopic level were cytologically indistinguishable between the myasthenic and normal immunoglobulin‐treated embryos. Myoblasts fused to form myotubes. which further differentiated into mature myofibers at the same time in both groups. Numerous neuromuscular junctions of normal ultrastructure and nerve fibers with myelin wrappings were observed in both cases. It is concluded that the increased neuron survival following myasthenic immunoglobulin treatment does not accompany retardation in differentiation and maturation of the target muscle which is contrary to the results obtained from studies utilizing neuromuscular blocking agents producing increased cell survival.
Morphological and biochemical maturation of rat astroglial cells grown in a chemically defined medium: Influence of an astroglial growth factor
Authors: Weibel M. , Pettmann B. , Labourdette G. , Miehe M. , Bock E. , Sensenbrenner M.
Published: April 2003

Astroglial cells from cerebral hemispheres of newborn rats were cultured for 5 days in Waymouth's MD 705/1 medium containing 10% fetal calf serum. Thereafter, cells were grown in a chemically defined medium consisting of basal Waymouth's medium supplemented with insulin (5 μg/ml) and fatty acid free bovine serum albumin (0.5 mg/ml). The cells underwent morphological and biochemical development over a period of 28 days. The changes in the amount of glial fibrillary acidic protein indicated a development of gliofilaments. The level of S100 protein increased during the entire culture period, while glutamine synthetase activity remained low and relatively constant. The addition of an astroglial growth factor, partially purified from bovine brain soluble extract, stimulated the morphological maturation of the astroglial cells. The cells extended cytoplasmic processes and resembled mature astrocytes. At the ultrastructural level an increase in free ribosomes was observed and the intermediate filaments became organized into large bundles. The amount of glial fibrillary acidic protein was not significantly increased, but the level of S100 protein and the glutamine synthetase activity were greatly enhanced. Our results indicate that astroglial cells undergo limited maturation in the chemically defined medium and that this process is positively affected by the astroglial growth factor.
Gangliosides induce microfilament‐dependent changes in membrane surface activity of Neuro‐2a neuroblastoma cells
Authors: Spero David A. , Roisen Fred J.
Published: April 2003

Exogenous mixtures of bovine brain gangliosides (BBG) initiated a rapid series of sequential changes in surface activity in Neuro‐2a neuroblastoma cells. These changes were characterized by the formation of microvilli, ridge‐like projections and ruffled membranes within l min of BBG exposure. A maximum number of microvilli was reached after 10 min exposure and disappeared by 30 min. Ruffled membranes were most prominent within 5 min, but were still present after 60 min. These surface changes were accompanied by the formation of a thick subcortical meshwork of microfilaments which formed the cores of the microvilli and ruffled membranes. The simultaneous treatment of cells with BBG and an agent known to disrupt microfilaments (cytochalasin D) inhibited the formation of both microvilli and ruffles. In contrast, the simultaneous application of BBG and agents affecting microtubule polymerization (Colcemid and taxol) did not inhibit the formation of these surface structures. Treatment of Neuro‐2a cells with cyclic AMP (cAMP) analogs (dibutyryl cAMP and 8‐bromo cAMP) induced a similar series of surface changes which were inhibited by cytochalasin D. Furthermore, these surface changes may be specific to cAMP since neither cGMP analogs (dibutyryl cGMP and 8‐bromo cGMP) nor 5′AMP induced surface changes. We propose that the initial membrane response to exogenous gangliosides is mediated by a microfilament‐dependent mechanism. The basic similarities in surface responses elicited by BBG and cAMP analogs suggest that the effects of gangliosides on surface activity may be induced in part by a cAMP‐dependent mechanism.
New formation of sensory cells in the tuberous organ (Electroreceptor) of Brienomyrus niger (Mormyridae) induced by transection of afferent nerve
Authors: Denizot J.P. , Libouban S.
Published: April 2003

The tuberous organs—cutaneous electroreceptors of the mormyrid fish Brienomyrus niger—were examined, with the light and electron microscope, after sectioning of the afferent nerve of the lateral line. Transection of the afferent nerve leads to the concomitant complete degeneration of all sensory cells, and to a differentiation of new sensory cells from accessory cells, which constitute the platform. Ultrastructural examination of the newly formed sensory cells shows that within a few days these gain the characteristics of normal sensory cells. The rapidly growing cytoplasm is enclosed in a folded membrane; the foldings develop typical dense microvilli. At the same time, in spite of lack of innervation, synaptic bars surrounded by vesicles differentiate in the cytoplasm; these are opposed to the basal cell membrane which is attached to the accessory cell platform. The newly formed sensory cells never reach the size of normally developed sensory cells. Their existence is transitory, for they degenerate, together with the sensory cells, one month after de‐afferentiation.
Membrane acetylcholinesterase in murine muscular dystrophy In vivo and in cultured myotubes
Authors: Poiana G. , Scarsella G. , Biagioni S. , Senni M.I. , Cossu G.
Published: April 2003

Murine muscular dystrophy is characterized by a reduction of the 10S molecular form of acetylcholinesterase (AChE); this reduction occurs in both strains of dystrophic mice and at the time of the phenotypic appearance of the disease.

In the present study we have analyzed the biochemical features, the cellular distribution and the developmental appearance of the AChE alteration. Sequential extractions with low salt, detergent and high salt revealed that this alteration affects only membrane‐bound forms (those requiring Triton X‐100 for solubilization), while both the low salt soluble and the high salt soluble forms appeared almost identical in normal and dystrophic muscles. Specific activity, sensitivity to different ions, pH dependence and Km were found to be identical in the enzymes from normal and dystrophic muscles, suggesting that the catalytic site of the 10S form is probably not altered.

Further analysis, by non‐denaturing gel electrophoresis, of the detergent soluble forms separated by sedimentation, revealed a single band for the 4S, a doublet for the 6S and three bands for the 10S peaks, indicating the existence of charge heterogeneity in AChE molecular forms. The corresponding molecular forms from dystrophic muscles behaved identically upon electrophoresis: the residual activity in the detergent soluble 10S form could still be separated into three bands, comigrating with their normal counterparts. Neuraminidase treatment resulted in a reduction of migration of both the 6S and 10S derived bands, but not of the 4S species, showing that sialic acid is added only to polymeric forms. Interestingly, the reduction of the 10S form appears to be linked to a developmental stage not reached in cell cultures, as cultured myotubes from muscles of dystrophic mice contained normal amounts of membrane‐bound AChE forms. The molecular mechanism underlying the reduction of the tetrameric membrane bound AChE form in dystrophic muscle and the possible functional consequences are discussed.
Distribution of pipecolic acid and proline in the developing rat brain and peripheral organs
Authors: Nishio Hiroaki , Yamada Yutaka , Giacobini Ezio , Segawa Tomio
Published: April 2003

The regional distribution of pipecolic acid and proline was studied in the adult and newborn rat brain. The brain concentration of pipecolic acid in the 1 and 3‐day‐old rat was 6–10 times higher than in the adult. The brain concentration of proline was found to be about 3 times higher in the brain of a newborn rat than in the adult, then it gradually decreased during the period of lactation. In plasma as well as in peripheral tissues, such as heart, kidney and liver, the highest concentration of pipecolic acid was observed at 1 day and in the adult. Secretion of pipecolic acid into the urine was highest at early stages of development. In the gastrointestinal tract, pipecolic acid was found to be most abundant in the adult, whereas low levels were found during the period of lactation. The developmental and regional changes in concentration of proline were quite different from those of pipecolic acid. Our results suggest that the contribution of dietary pipecolic acid in building up levels of pipecolic acid in rat brain is small.
Perinatal dietary exposure to soy lecithin: Altered sensitivity to central cholinergic stimulation
Authors: Bell Joanne M. , Whitmore William L. , Barnes Gloria , Seidler Frederic J. , Slotkin Theodore A.
Published: April 2003

The effects of perinatal exposure to soy lecithin preparation (SLP) on the development of cholinergic responses in the rat brain were examined by assessing the ability of intracisternally administered carbachol to stimulate 33Pi incorporation into phospholipids in vivo, an effect of carbachol mediated by muscarinic cholinergic receptors. Maternal intake of SLP produced a suppression of the cholinergic response in the offspring, an effect which was specific in that basal (unstimulated) incorporation rates were not reduced (in fact, they eventually became elevated), nor was the response to another neurotransmitter (dopamine) compromised. The effect occurred early in the preweanling stage, a period in which SLP exposure also enhances development of cholinergic nerve terminals. These results suggest that SLP exposure has a major effect on cholinergic synaptic development and reactivity, followed by secondary changes in other neurotransmitter pathways and by more generalized effects on basal membrane phospholipid turnover.
Increase in neuronotrophic activity during the period of smooth muscle innervation
Authors: Rush R. A. , Abrahamson I. K. , Murdoch S. Y. , Renton F. J. , Wilson P. A.
Published: April 2003

The expansor secundariorum is a unique smooth muscle of the avian wing that receives a dense sympathetic innervation and contains high concentrations of survival factors for sympathetic neurons. In the present study it has been possible to simultaneously examine the appearance of the neuronotrophic activity and the arrival of nerve fibres during the period of innervation. The results show that catecholamine containing nerve fibres can first be detected within the muscle on the fourteenth day of incubation (stage 40) followed by a rapid increase in the density of fibres during the next few days until the adult pattern is reached shortly before hatch. Biochemical estimation of the innervation process by measurement of dopamine β‐hydroxylase activity was supported by the histochemical findings. Estimation of neuronotrophic activity revealed that muscle from stage 40 embryos contains only low levels of activity which increases rapidly as innervation proceeds and further, that this increase in neuronotrophic activity was directly correlated with the dopamine β‐hydroxylase activities. Possible mechanisms regulating this dramatic increase in the specific activity of trophic factors are discussed.
The biphasic effect of phospholipase A2 inhibitors on axon elongation
Authors: Suburo Angela , de Job Claudia Cei
Published: April 2003

We have analyzed changes in axon elongation rate in vitro induced by three different phospholipase A2 inhibitors, namely, bromophenacyl bromide (BPB), quinacrine and Sr2+. Spinal ganglia were obtained from 19.5‐day‐old rat fetuses and explanted onto polyornithine substrata. Axon length was measured at 1, 2 and 3 days in vitro (d.i.v.). We have previously shown that BPB added at 5 hr in vitro (h.i.v.) modifies the structure of growth cones and inhibits or promotes axon elongation according to its concentration. Now, we have observed that 0.5 × 10−6 M BPB also stimulates axon elongation when added at 1 d.i.v. Culture media with different Ca2+ concentrations were used to test Sr2+ added at 1 d.i.v. In 2.3 mM Ca2+ only an inhibitory effect was observed with 8 mM Sr2+. On the other hand, in 0.3 mM Ca2+, axon growth was stimulated by 0.6–1.2 mM Sr2+ but inhibited by 6 mM Sr2+. Quinacrine, added at 5 h.i.v. was inhibitory at 10−5 M and showed no effects at 10−6 M. However, after washing quinacrine, normal elongation rate was recovered by those previously treated with 10−5 M and axon growth was enhanced in those treated with 10−6 M. Since three different phospholipase A2 inhibitors, tested in different situations, produce a similar biphasic effect on axon elongation rate, we postulate that this enzymatic activity is involved in the motility of axon growth cones.
Thyroidal induction of tubulin in brain development — identification of the target cell
Authors: Chatterjee D. , Sarkar P.K.
Published: April 2003

Exposure of organ cultures of newborn rat brains to tri‐iodothyronine (T3) followed by cell fractionation as well as direct exposure of prefractionated neuronal (N) and glial (G) cells to the hormone results in an almost selective induction of tubulin in the glial cells. This is established from two independent assays of tubulin, viz. colchicine binding and vinblastin precipitation. In the newborn rat brain, the tubulin content of the G cells is almost 3‐fold higher than that of the N cells. Treatment with T3 elicits 40–50% stimulation of tubulin in the G cells within 2 hr without any significant increase in the N cells. Brains from 8‐ or 50‐day‐old rats are irresponsive to induction to tubulin by T3. The rate of incorporation of [3H]leucine into total protein is very similar in both N and G cells of newborn rat brain but that into tubulin of G cells is about 3‐fold higher than that of N cells. T3 promotes this incorporation by over 30% in the G cells with only a marginal 5% increase in the N cells. The overall results suggest that the glial cells represent the target cells for the T3‐induced synthesis of tubulin, the major structural protein of the developing brain.
Innervation is required to stabilize and amplify creatine kinase activity in regenerated extensor digitorum longus muscles of rats
Authors: Rossi Anna Maria , Savarese Nicola , Cotrufo Roberto
Published: April 2003

Neural control of creatine kinase (CK, adenosine 5′‐triphosphate creatine phosphotransferase: EC 2.7.3.2) was investigated by measuring enzymatic activity and isoenzymatic representation of CK in extensor digitorum longus (EDL) muscles of adult rats during and after regeneration. Experimental models were ischemized EDLs, reversibly or permanently denervated. Results showed that, during regeneration, CK in muscle fibers was likewise modified in both reversibly and permanently denervated EDLs. After regeneration a clear dichotomy was observed between the regenerated EDLs which were innervated and recovering CK activity, and those in which innervation was prevented and were rapidly losing activity. Further to investigate the neural influence on CK turnover, merely denervated agematched EDLs were analysed and found to lose CK activity rapidly. The major conclusions are that during regeneration muscle CK is autonomously expressed, but following regeneration neural influence becomes an absolute requirement for stabilization and amplification of CK.
Thrombin is a potent mitogen for rat astroblasts but not for oligodendroblasts and neuroblasts in primary culture
Authors: Perraud Frédéric , Besnard François , Sensenbrenner Monique , Labourdette Gérard
Published: April 2003

Astroblasts from brain of newborn rat can survive and even proliferate to some extent in a chemically defined medium containing no other growth factor than insulin, providing they are grown first in the presence of fetal calf serum for at least 4 days (Weibel et al., 1984, Int. J. devl Neurosci, 2, 355–366). We found that thrombin is a potent mitogen for these cells, in vitro. The mitogenic activity of thrombin for astroblasts can be compared to that of the astroglial growth factor 2 (AGF2), also known as basic fibroblast growth factor (bFGF), which was, up to now, the most active factor on astroblasts. However, in contrast to the bFGF, thrombin does not modify significantly the morphology of the cells and their synthesis of glutamine synthetase, an astroglial marker in rat brain. Some other proteases are also able to stimulate the proliferation of astroblasts, but to a lesser extent than thrombin. Thrombin does not stimulate the proliferation of oligodendroblasts from newborn rat and of neuroblasts from 13‐day‐old rat embryo. These results suggest that in the central nervous system thrombin might play a role in the induction of astrocyte proliferation after brain injury.
Detachment of cultured neurons from substratum by astrocytic membrane‐associated activity. Implications for CNS regeneration?
Authors: Gilad Gad M. , Gilad Varda H.
Published: April 2003

In primary cultures of dissociated cells from 6‐day‐old rat cerebellum, astrocytes proliferated and grew under the initially formed network of neurons. The growing astrocytes invaded the culture growth surface, while the neurons detached from the substratum and died. By 8 days after plating a confluent layer of astrocytes has taken over the entire growth surface area and neurons have disappeared. Membrane fractions prepared from astrocytes of 8‐day‐old cultures, have led to detachment of growing neurons. After 2 days in the presence of membrane fractions, about 70% of the neurons disappeared and many of the remaining ones had detached or retracted processes. The results indicate that the well‐known phenomenon of disappearance of neurons from mixed primary CNS cultures, is probably caused by a membrane‐associated activity of the proliferating astrocytes which can disturb neuron‐substratum attachments. Our in vitro study implies that astrocytes may have a membrane‐associated activity which is expressed under certain conditions and can disturb the contacts of growing or regenerating axons with their growth substrata.
Sex steroids promote neurite growth in mesencephalic tyrosine hydroxylase immunoreactive neurons in vitro
Authors: Reisert Ingrid , Han Victor , Lieth Erich , Toran‐Allerand Dominique , Pilgrim Christoph , Lauder Jean
Published: April 2003

The influence of steroid hormones on the differentiation of catecholaminergic and serotonergic (5‐HT) neurons was studied in dissociated cell cultures from embryonic day 14 (E14) rat diencephalon, mesencephalon and metencephalon treated for 6 days with 17β‐estradiol (E), testosterone (T), 5α‐dihydrotestosterone (DHT), progesterone (P), dexamethasone (DEX), or E + T. The effects of these hormones on morphologic differentation were determined by morphometric measurements of total length of neurites of immunocytochemically identified neurons in culture, which were stained with antisera against tyrosine hydroxylase (TH) or 5‐HT. A significant increase in neurite length was observed in cultures of TH‐immunoreactive (TH‐IR) neurons from the mesencephalon treated with E, T, E + T, but not with P, DHT or DEX. Based on labeling with [3H]dopamine (DA) uptake and competition with specific inhibitors, these mesencephalic TH‐IR cells appear to represent DA neurons of the A8–A10 groups (which includes the substantia nigra). No statistically significant effects of these steroids were observed on TH‐IR neurons from the diencephalon (assumed to be precursors of the tuberoinfundibular and incertohypothalamic dopaminergic groups). The 5‐HT neurons of the raphe nuclei (metencephalon) showed no statistically significant response to steroids. We conclude that during the early fetal period, sex steroids can affect the morphologic differentiation of mesencephalic DA neurons in vitro, indicating that these hormones are capable of selectively influencing the development of a specific population of monoamine neurons during this critical period.
Plasminogen activator secretion in relation to Schwann cell activities
Authors: Krystosek Alphonse , Verrall Shahla , Seeds Nicholas W.
Published: April 2003

The molecular characterization of neural secretory plasminogen activators and their possible association with two well‐defined Schwann cell activities (mitosis and migration) were investigated. Schwann cells from peripheral sensory ganglion roots were established as primary cultures of high purity. Conditioned culture medium was fractionated by gel electrophoresis and assayed for plasminogendependent in situ caseinolytic activity. Both tissue‐type and urokinase‐type plasminogen activators were detected early in culture. The amount of the tissue‐type form decreased with increasing time in culture. Proliferative ability measured as [3H]thymidine incorporation into the nucleus varied under several culture conditions and was lowest in defined serum‐free medium; however, these serum‐free cultures expressed the highest level of plasminogen activator secreting cells. Combined autoradiography/fibrin overlay assays allowed for a direct analysis at the single cell level; interestingly the findings did not support the idea of an obligatory expression of the enzyme in replicating cells. Cell motility was assayed in combined gold particle clearing/fibrin overlay assays and likewise no obligatory association of protease secretion and cell movement could be demonstrated. The results are discussed in relation to previous tudies in this field.
Ontogeny of the opiate phenotype: An approach to defining transsynaptic mechanisms at the molecular level in the rat adrenal medulla
Authors: La Gamma E. F. , De Cristofaro J. D. , Agarwal B. L. , Weisinger G.
Published: April 2003

Transmitter phenotypic expressions is a dynamic cellular process governed by multiple interactions with the neuronal environment. During sympathoadrenal development the arrival of presynaptic nerve terminals at the adrenal chromaffin cell (in the immediate postnatal period), coincides with the acquisition and subsequent development of a variety of transmitter biosynthetic capacities. Data discussed herein supports the contention that synaptic connections serve a central role in triggering the ontological cascade. Disruption of the normal timing of innervation events is detrimental to subsequent function and results in permanent deficiencies in development. In addition, alteration of transmitter biosynthetic regulatory mechanisms appears to reside at the level of gene expression. In view of this, additional molecular approaches are necessary to further elucidate the fundamental basis of neuronal transmitter phenotypic plasticity. Our approach to this problem represents a logical extension of previous research in this area and ultimately, will involve characterizing transcription activator molecules important in transmitter gene expression at various ontological ages.
Co‐localization and plasticity of transmitters in peripheral autonomic and sensory neurons
Authors: Morris J. L. , Gibbins I. L.
Published: April 2003

Immunohistochemical studies have shown that most peripheral autonomic and sensory ganglia are heterogeneous, consisting of several populations of neurons which can be distinguished by their content of peptide and non‐peptide transmitters and transmitter‐associated enzymes. Many neurons contain several different potential transmitters, especially neuropeptides. Some neuropeptides have been localized in more than one population of autonomic and sensory neurons. However, the peptide often occurs together with a distinctive combination of additional transmitters in each neuronal class. The precise combination of transmitters found in any individual neuron is highly correlated with the peripheral target of the neuron. This indicates that immunohistochemically defined neuronal populations represent distinct functional classes of neurons. In an increasing number of cases, many of the potential transmitters contained in a particular neuron have been shown to be released from the nerve terminals, and to contribute to presynaptic or postsynaptic effects of nerve activation. Despite this association between the combination of potential transmitters contained in a neuron, and the function of the neuron, not all transmitters or transmitter‐associated enzymes are expressed equally at all times in the life of a neuron: the levels of some substances change dramatically during development; some are detected only after experimental alteration of the environment of the developing or mature neurons. Taken together, these results indicate that, during development, pathway‐specific information influences the differentiation of peripheral autonomic and sensory neurons. Furthermore, the expression of neuropeptides and transmitter‐associated enzymes in a particular neuron appears to be under continuous regulation. These phenomena demonstrate the complexity and precision involved in development and maintenance of the peripheral autonomic and sensory nervous systems.
Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro
Authors: Verna Jean‐Marc , Fichard Agnes , Saxod Raymond
Published: April 2003

The neuritic growth patterns obtained on substrates made of several glycosaminoglycans (GAGs) bound to type I collagen were analysed and compared in primary cultures of chick embryo dorsal root ganglion grown in serum‐free supplemented medium. In 2‐day cultures grown on type I collagen or heparan sulphate (HS)‐ collagen surfaces, ganglionic expiants exhibit a dense, symmetrical network of long, parallel neuritic processes and very few flat migrating non‐neuronal cells. In contrast, on either dermatan sulphate (DS), chondroitin‐6‐sulphate (C6S) or hyaluronic acid (HA)‐bound collagen substrates, neurons form irregular nerve fibre patterns; indeed, neuntes follow convoluted paths and often, after abrupt turns, totally reverse their direction of extension. Experiments were carried out in which a choice was given to growing neural processes between collagen or GAG‐collagen substrates. While growth cones elongating over type I collagen easily cross the border with HS‐bound collagen surface and indiscriminately extend on this substrate, in contrast, neurites generally avoid surfaces coated with DS, C6S or HA and change their direction of growth in order to stay on collagen. The binding of DS, C6S or HA, but not HS, to type I collagen thus decreases its ability to promote neurite elongation. The interaction of neuronal cells with these extracellular matrix components by restricting neurites in their paths of extension may, therefore, play a role in the patterning of the nervous circuitry.
Characterization of resting membrane potential and its electrogenic pump component in cultured chick myotubes
Authors: Brodie Chaya , Sampson S. R.
Published: April 2003

The role of the electrogenic Na+‐K+ pump in the determination of the level of the resting membrane potential in cultured chick limb muscle was investigated. Transmembrane resting potential and ouabain‐sensitive86Rb‐uptake were measured in myotubes at different ages in culture from 2 to 10 daysin vitro. Inhibition of the Na+‐K+ pump with ouabain prevented the developmental increase in membrane potential which normally follows fusion of myotubes (day 2–3). In mature myotubes, ouabain caused a dose‐related decrease in both membrane potential and86Rb‐uptake, with values for EC50 and maximal effect being nearly the same on both variables. The decrease in membrane potential by ouabain, up to 20 mV maximum, occurred within 2–5 sec and was not accompanied by detectable changes in input resistance. Membrane potential was also reduced by a decrease in temperature of the recording medium and removal of extracellular K+, both of which reduce Na+‐K+ pump activity. We also found that the relation between membrane potential and extracellular K+ concentration was completely attenuated by ouabain in the physiological range (2–10 mM). We conclude that the electrogenic Na+‐K+ pump plays an important role in the determination of the resting membrane potential of chick myotubes and that regulation of its level is not entirely explained by the diffusion potential hypothesis.
Glial‐conditioned medium and attachment to ConA are essential for long‐term culture of cortical neurons
Authors: Bauer H. C. , Tontsch U.
Published: April 2003

Cortical brain cells from 14‐day‐old mouse embryos were seeded on various substrates and cultivated in serum‐free medium with or without conditioned medium from astrocytes or C6 glioma cells. Poly‐l‐lysine was shown to be the best substrate for cell attachment followed by Concanavalin A (ConA) and adhesion particles derived from glia cells. Cells grown on ConA sprouted rapidly and formed large networks. Survival of neurons was greatly prolonged when glia‐conditioned medium (GCM) was present in the culture medium. Cells grown on ConA were then viable for more than 4 weeks. Without GCM, neurons survived in culture for about 2 weeks, regardless of the substrate. Endothelial cell growth supplement or acidic fibroblast growth factor increased survival of neurons but also stimulated proliferation of astrocytes.
Calcium and calmodulin levels in late embryonic and early hatched japanese quail brain
Authors: Alston‐Mills Brenda , Shankarappa Basavaraju , Douglass Larry
Published: April 2003

Characterizing the last phases of embryonic avian brain development are increased brain activity and increased absorption of shell calcium. The calcium‐binding protein, calmodulin, regulates many activities of calcium. In neural tissue, calmodulin modulates neural transmission, and is required for the phosphorylation of synaptosomal proteins. Therefore, the objective was to compare levels of brain calcium and calmodulin in the Japanese quail. Brain extracts from embryonic days 11, 15, hatch, and 5 days post‐hatch (n = 7/group) were analysed for calcium, protein and calmodulin. Despite increases in protein between embryonic ( = 0.126 and 0.145 mg/mg wet wt) and hatched groups ( = 0.183 and 0.221 mg), no significant increases in calmodulin were observed (237–279 ng/mg protein). Calcium levels in the brain were U‐shaped with low levels at embryonic day 1 (341 μg/mg wet wt) and post‐hatch day 5 (315 μg/mg wet wt) with higher levels on embryonic day 15 (425 μg/mg wet wt) and at hatch (433 μg/mg wet wt). Calmodulin levels do not show a developmental pattern similar to calcium and protein levels or with reports of brain activity.