Differences in growth of neurons from normal and regenerated teleost spinal cord in vitro

Summary Explants and dissociated cells from normal adult spinal cord and regenerating cord of the teleostApteronotus albifrons were grown in vitro for periods of 8 to 12 wk. During this time the neurons showed extensive neurite outgrowth. Neurite outgrowth from tissue explants and dissociated cells of regenerated spinal cord starts sooner and is more profuse than that from normal (unregenerated) cord. Neurite outgrowth is maximized by using adhesive substrata and a high density of explants or dissociated cells. Inasmuch asApteronotus does regenerate its spinal cord naturally after injury, whereas mammals do not, this culture system will be useful to study factors that control (permit) regeneration of spinal neurons in this adult vertebrate.     

Protective effect of hypothermia during ischemia in neural cell cultures

Hypothermia offers protection from the effects of ischemia in small animals. We have recently shown that similar to small animals, hypothermia may also be protective in an astrocytic model of simulated ischemia in cell culture. This study was designed to look at the protective effects of hypothermia in cultures of cerebellar granular (glutamatergic) and cortical (GABAergic) neurons. We used LDH release into the medium as an indicator for neuron damage. Experiments were all done in sister cultures, in groups of six cultures at two temperatures (37 and 32 degrees Celsius). The duration of ischemia was three hours in cerebellar granular neuronal cell cultures and six hours in cortical neurons. LDH release was measured immediately after the insult. Hypothermia protected both granular and cortical neurons. In granular cells, LDH release was 62+/–18 at 32 degrees and 212+/–15 at 37 degrees (p=0.02). Cortical neurons showed LDH release of 15+/–2 at 32 degrees and 32+/–2 at 37 degrees (p=0.005). Our study suggests that similar to astrocytes, the protective effects of hypothermia are evident in neuronal cell cultures from the cerebellum and the cerebral cortex. Cell culture systems should prove useful techniques in understanding mechanisms of hypothermic protection during simulated ischemia in neurons from different sites.     

Uptake and metabolism of glutamate and aspartate by astroglial and neuronal preparations of rat cerebellum

Astrocytes, neuronal perikarya and synaptosomes were prepared from rat cerebellum. Kinetics of high and low affinity uptake systems of glutamate and aspartate, nominal rates of¹⁴CO₂ production from [U–¹⁴C]glutamate, [U–¹⁴C]aspartate and [1–¹⁴C]glutamate and activities of enzymes of glutamate metabolism were studied in these preparations. The rate of uptake and the nomial rate of production of¹⁴CO₂ from these amino acids was higher in the astroglia than neuronal perikarya and synaptosomes. Activities of glutamine synthetase and glutamate dehydrogenase were higher in astrocytes than in neuronal perikarya and synaptosomes. Activities of glutaminase and glutamic acid decarboxylase were observed to be highest in neuronal perikarya and synaptosomes respectively. These results are in agreement with the postulates of theory of metabolic compartmentation of glutamate while others (presence of glutaminase in astrocytes and glutamine synthetase in synaptosomes) are not. Results of this study also indicated that (i) at high extracellular concentrations, glutamate/aspartate uptake may be predominantly into astrocytes while at low extracellular concentrations, it would be into neurons (ii) production of -ketoglutarate from glutamate is chiefly by way of transamination but not by oxidative deamination in these three preparations and (iii) there are topographical differences glutamate metabolism within the neurons.     

动态神经网络中的同步振荡

目前有一种假设认为同一视觉对象是由一群神经元的同步振荡活动来表征的。这一神经元发放活动的时间特性,是解决视觉信息处理中“结合问题（Ｂｉｎｄｉｎｇｐｒｏｂｌｅｍ）”的可能机制。本文用我们所提出的一种简化现实性神经网络模型［１］所构造的时滞非线性振子网络［２］,模拟生物神经网络的同步振荡活动。并考虑了振子各参数的设置与振荡活动的关系,以及网络振子间耦联对同步活动的影响．     

In vitro peptidergic neurons from the adult locust pars intercerebralis: Morphological and immunocytological studies

Primary cell cultures were prepared from a major neurosecretory center of the adult locust brain, the pars intercerebralis, in order to characterize neurosecretory cells growingin vitro. Individual pars intercerebralis could be removed free of surrounding tissue and dissociated by mechanical treatment. Mature neurosecretory neurons of different sizes regenerate new neurites during the initial three daysin vitro in serum-free medium. They show a tendency to sprout one primary neurite from which fine processes develop. By means of electron microscopy, we observed the integrity of the cellular organelles, indicating that cultured neurons are healthy, and we were able to distinguish three types of neurosecretory neurons on the basis of the ultrastructural aspects of the neurosecretory material. These three types have the same ultrastructural characteristics asin situ neuroparsin, ovary maturing parsin and locust insulin related peptide neurons. Immunogold labelling at the electron microscopic level, using the two available specific antibodies, anti-neuroparsin and anti-ovary maturing parsin, confirms the morphological characterization of neuroparsin and ovary maturing parsin cells. These results show for the first time that cultured locust neurosecretory neurons behave like thosein vivo, in terms of their ultrastructure and immunocytochemistry. Moreover, the presence of recently-formed neurosecretory material both in the Golgi zone of the perikaryon and in the neuronal processes indicates that cultured neurons have functional capacity since they are able to synthesizede novo and to transport the neurosecretory material along the neurite. Thus our well-characterized culture system provides a suitable invitro model to investigate the secretory mechanism of locust neurosecretory neurons.     

Presynaptic Nicotinic Receptors Stimulate Increases in Intraterminal Calcium of Chick Sympathetic Neurons in Culture

Abstract: Stimulation of chick sympathetic neurons in culture by the cholinergic agonists acetylcholine, nicotine, and 1,1-dimethyl-4-phenylpiperazinium (all at 10–1,000 µmol/L) induced concentration-dependent increases of free calcium levels measured by fura 2 fluorescence in neuronal processes. The response evoked by acetylcholine had both nicotinic and muscarinic components, whereas that induced by 1,1-dimethyl-4-phenylpiperazinium was purely nicotinic. Tetrodotoxin (0.3 µmol/L) blocked completely the increase of intraterminal free calcium level evoked by electrical stimulation. On the other hand, stimulation with 1,1-dimethyl-4-phenylpiperazinium still evoked 20–25% of the control response in the presence of tetrodotoxin. The concentration-response relationship of 1,1-dimethyl-4-phenylpiperazinium stimulation did not differ in the absence and in the presence of tetrodotoxin. The nicotinic antagonists d -tubocurarine (10 µmol/L) and mecamylamine (10 µmol/L), but not α-bungarotoxin (125 nmol/L), prevented the increase of intraterminal free calcium level evoked by 1,1-dimethyl-4-phenylpiperazinium (100 µmol/L) in the presence of tetrodotoxin. These observations indicate the presence of nicotinic receptors on neuronal processes that increase the intraterminal concentration of free calcium and probably modulate transmitter release. Their pharmacological properties are similar to those of nicotinic receptors located on neuronal cell bodies.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

Neurotrophic Effects of l-DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures

Abstract: l -DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of l -DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. l -DOPA (50 µ M ) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where l -DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 µ M l -DOPA. The stereoisomer d -DOPA (50–400 µ M ) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 µ M ) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of l -DOPA is stereospecific but independent of the production of dopamine. However, l -DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by l -buthionine sulfoximine (3 µ M for 24 h) blocked the neurotrophic action of L-DOPA. N -Acetyl- l -cysteine (250 µ M for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of l -DOPA. These data suggest that the neurotrophic effect of l -DOPA may be mediated, at least in part, by elevation of glutathione content.