Trophic effect of beta-amyloid precursor protein on cerebral cortical neurons in culture

We investigated the effect of human beta-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons.     

Intranuclear localization of iron in neurons of mammalian brain

Using Perls’ histochemical method, iron was revealed in the brain structures of human and rat. Iron accumulation was observed in perivascular areas and neuropil of substantia nigra as well as in white matter of cerebellum. After diaminobenzidine enhancement of histochemical reaction, the iron was revealed in nucleoli of many neurons, which is described for the first time in animal cells.     

Dendritic spines of developing rat cortical neurons in culture

The formation of spines and their association with synapses were examined in developing cultured rat cortical neurons using fluorescence labeling techniques. Small protrusions were found on the processes of cultured cortical neurons after seven days in vitro (DIV), and the density of protrusions almost halved during the second week in vitro, after which it remained unchanged throughout the third week in vitro. The proportion of protrusions associated with the accumulation of the presynaptic marker, synaptophysin, increased steadily from <5% at 7 DIV to approximately 50% at 21 DIV. Based on the absence or presence of an enlargement at the end, protrusions on processes were further divided into filopodia and spines, respectively. The percentage of protrusions that were classified as spines increased steadily from approximately 5% at 3-4 DIV to approximately 80% at 18-20 DIV. The percentage of spines associated with synaptophysin accumulation increased gradually as the cortical neurons developed in vitro, reaching a plateau of approximately 40% after two weeks. However, the percentage of filopodia associated with synaptophysin accumulation never exceeded 5% during the first three weeks in vitro. Double-label staining the microfilaments and beta-tubulin or phosphorylated neurofilament H of cultured neurons further revealed many spines without any nearby axon-like processes. These findings suggest that spines are the dominant form of protrusion on the processes of more mature cortical neurons, that spines are the preferential sites where synapses reside, and that maintaining constant contact with axons is not essential for the formation of spines in cultured cortical neurons.     

The dynamics of a neuronal culture of dissociated cortical neurons of neonatal rats

Neuronal networks of dissociated cortical neurons from neonatal rats were cultured over a multielectrode dish with 64 active sites, which were used both for recording the electrical activity and for stimulation. After about 4 weeks of culture, a dense network of neurons had developed and their electrical activity was studied. When a brief voltage pulse was applied to one extracellular electrode, a clear electrical response was evoked over almost the entire network. When a strong voltage pulse was used, the response was composed of an early phase, terminating within 25 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase occurred with a precise timing with a small jitter and the electrical activity initiated by a localized stimulation diffused significantly over the network. In contrast, the late phase was characterized by the occurrence of clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by adding small amounts of d(−)-2-amino-5-phosphonovaleric acid to the extracellular medium, or by increasing the amount of extracellular Mg²⁺. The electrical activity of the network was substantially increased by the addition of bicuculline to the extracellular medium. The results presented here show that the neuronal network may exist in two different dynamical states: one state in which the neuronal network behaves as a non-chaotic deterministic system and another state where the system exhibits large spatio-temporal fluctuations, characteristic of stochastic or chaotic systems. Received: 8 June 1999 / Accepted in revised form: 10 January 2000     

A fatal effect of hornet venom on rat-brain cortical neurons

In a previous study, it was shown that the hornet venom or, more specifically, its venom sac extract (VSE) possesses deoxyribonuclease activity that exerts an effect both on insects as well as on mammals. We have now examined the effect of hornet VSE on primary culture of rat cortical neurons. Judging on the basis of our results, VSE induces a rapid cell death by a) permeabilizing the cell membrane, b) inducing DNA breaks, and c) cleaving the nuclear protein poly-ADP-ribose polymerase (PARP-1), thereby preventing DNA repair.     

Concurrent acetylcholinesterase staining and gamma-aminobutyric acid uptake of cortical neurons in culture

Gamma-aminobutyric acid (GABA) uptake and acetylcholinesterase (AChE) content were demonstrated concurrently in cortical neurons grown in tissue culture. Positive reactions either for GABA uptake or for AChE content were encountered in pyramidal and stellate, as well as spindle-shaped neurons. Neither reaction was confined to a specific morphological subtype. Nearly half the neurons were negative for either reaction. Most of the remaining neurons were positive only for GABA or only for AChE. However, a subpopulation of neurons showed not only a high AChE content, but also an avid GABA uptake. Thus, four types of neurons could be identified on the basis of these two reactions. The high AChE content in some of the cortical neurons that also showed GABA uptake indicates that there are at least two distinct types of GABAergic neurons.     

The effect of ligands on the uptake of iron by cells in culture.

Uptake of iron by a mammalian epithelial cell line (CNCM I-221) was shown to be dependent on the nature of the iron complex. Iron uptake was demonstrated by cytochemical staining and determination of redox-reactive iron in cell lysates. Three classes of ligands were investigated: (i) low molecular weight hydrophilic compounds, represented by ethylenediamine-tetraacetic acid (EDTA) and other charged ligands such as adenosine phosphates (ATP, ADP, AMP) and diethylenetriaminepentaacetic acid (DTPA), (2) low-molecular weight lipophilic ligands such as 8-hydroxyquinoline (8-HQ) and (3) a high molecular mass ligand, dextran. Iron complexed to 8-HQ accumulated intracellularly, the uptake rate of iron being 4.16 fmoles cell-1 h-1 of exposure at 37 degrees C or 3.86 fmoles cell-1 h-1 at 4 degrees C. Iron-dextran was endocytosed and retained in phagosomes. The uptake rate of iron following exposure to iron dextrans was found to be 5.6 fmoles cell-1 h-1 of exposure at 37 degrees C. In contrast to iron/8-HQ, uptake of iron dextran by cells was inhibited at 4 degrees C. Iron complexed to low molecular weight hydrophilic ligands was not taken up by cells. Cytotoxicity was measured by reduction of plating efficiency or tritiated thymidine incorporation. These tests showed that toxic effects of added iron were demonstrable only in cells exposed to the complex with 8-HQ.     

The effect of the intracellular level of free iron on cell proliferation in culture

Dependence of changes in the intracellular free iron content upon cellular proliferation has been studied on mammalian cells in vitro. It has been established that picolic acid (PA)--a natural metal chelating agent of variable valency--inhibits the proliferation of cultivated pig embryo kidney cells (SPEV). Simultaneously the free iron quantity was decreased 2-fold as compared with the norm. PA block was removed by substituting PA-containing cultivated media for the PA-free one. It was accompanied by complete recovery of the free iron quantity in the cells. The regulation of cell proliferation is likely to correlate with the intracellular free iron content.     

Identification and characterization of a population of motile neurons in long-term cortical culture

The specific phenotypes and progression to maturity of primary cortical neurons in long-term culture correlate well with neurons in vivo. Utilizing a model of neuronal injury in long-term cultures at 21 days in vitro (DIV), we have identified a distinct population of neurons that translocate into the injury site. 5-bromo-2'-deoxyUridine (BrdU) incorporation studies demonstrated that neurons with the capacity to translocate were 21 days old. However, this motile ability is not consistent with the traditional view of the maturation and structural stability of neurons in long-term culture. Therefore, we examined the neurons' cytoskeletal profile using immunocytochemistry, to establish relative stage of maturation and phenotype. Expression of marker proteins including beta-III-tubulin, alpha-internexin, NF-L and NF-M, tau and L1 indicated the neurons were differentiated, and in some cases polarized. The neurons did not immunolabel with NF-H or MAP2, which might suggest they had not reached the level of maturity of other neurons in culture. They did not express the microtubule-associated migration marker doublecortin (DCX). Cytoskeletal disrupting agents were used to further investigate the role of the microtubule cytoskeleton in translocation, and microtubule destabilization significantly enhanced aspects of their motility. Finally, molecular guidance cues affected their motility in a similar manner to that reported for both axon guidance and early neuron migration. Therefore, this study has identified and characterized a population of motile neurons in vitro that have the capacity to migrate into a site of injury. These studies provide new information on the structurally dynamic features of subsets of neurons.     

Epileptiform postsynaptic currents in primary culture of rat cortical neurons: Calcium mechanisms

In this study we demonstrate that the primary culture of rat cortical neurons is a convenient model for investigations of epileptogenesis mechanisms and specifically, of the postsynaptic epileptiform currents (EC) reflecting periodical asynchronous glutamate release. In particular, we have revealed that in primary culture of cortical neurons EC can appear spontaneously or can be triggered by the withdrawal of magnesium block of NMDA receptor channels or by shutting down GABAergic inhibition. EC were found to depend on intracellular calcium oscillations. The secondary calcium release from intracellular stores was needed for EC synchronization. EC were suppressed by the influences causing either neuronal calcium overload or decrease of intracellular calcium concentration. Calcium entry into neurons in the case of NMDA receptor hyperactivation or in the case of calcium ionophore ionomycin treatment eliminated EC. The suppression of EC also occurred after a decrease of intracellular calcium concentration induced by BAPTA loaded into the neurons or by stimulation of calcium removal from cells via Na⁺/Ca²⁺ exchanger by 1 nM ouabain. Partial dependence of EC on action potential generation was found. Thus, EC in neurons are activated by intracellular periodic calcium waves within a limited concentration window.     

Neurotrophic effect of isoquinoline derivatives in primary cortical culture.

Recent studies indicate that the N-methyl-D-aspartate (NMDA) antagonist, (+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (FR 115427), enhanced neuronal survival in primary culture of cortical neurons from mouse embryos. In the present study isoquinoline derivatives were examined for the neurotrophic activity in primary culture of cortical neurons and were also examined for anti-NMDA activity. In spite of varying level of anti-NMDA activity, isoquinoline derivatives enhanced neuronal survival at the concentration of 10 microM. To elucidate of the mechanisms of neurotrophic activity in primary cortical culture, nicardipine and flunarizine, known calcium channel blockers, were also tested. Neither nicardipine nor flunarizine showed neurotrophic activity up to the doses causing toxicity in cultured neurons. NBQX, an AMPA receptor antagonist, was also tested for neurotrophic activity. However no enhancement of neuronal survival was observed. These data suggest that one of the mechanisms to promote neuronal survival may depend on the structure of isoquinoline ring. Moreover neurotrophic activity observed in our culture systems might not relate on anti-NMDA activity, blockade of voltage dependent L-type calcium channels and antagonization of AMPA receptor.     

Protective effect of panaxydol and panaxynol on sodium nitroprusside-induced apoptosis in cortical neurons

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. The protective effect of panaxydol (PND) and panaxynol (PNN) on sodium nitroprusside (SNP)-induced neuronal apoptosis and potential mechanism were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN for 24 h following 1 mM SNP, an exogenous NO donor, exposure for 1 h, resulted significantly in reduction of cell death induced by SNP determined by MTT assay, LDH release and Hoechst staining. 5 μM PND and PNN also reduced the up-regulation of the pro-apoptotic gene, Bax, down-regulation of the anti-apoptotic gene, Bcl-2. The observations demonstrated that PND and PNN protect neurons against SNP-induced apoptosis via regulating the apoptotic related genes. The results raise the possibility that PND and PNN reduce neurodegeneration in the Alzheimer's brain.     

The action potential in mammalian central neurons

The action potential of the squid giant axon is formed by just two voltage-dependent conductances in the cell membrane, yet mammalian central neurons typically express more than a dozen different types of voltage-dependent ion channels. This rich repertoire of channels allows neurons to encode information by generating action potentials with a wide range of shapes, frequencies and patterns. Recent work offers an increasingly detailed understanding of how the expression of particular channel types underlies the remarkably diverse firing behaviour of various types of neurons.     

The role of the thalamic ventrolateral nucleus in the cortical effect on the activity of vestibular neurons

Electrocoagulation of lateral vestibular nucleus (NVL) reduces inhibitory effect of the motor and somatosensory areas and enhances the inhibitory effect of limbic, vestibular, and orbital cortical areas. Facilitating effect was enhanced by electrostimulation of the motor area and reduced by the stimulation of other cortical areas. Following the coagulation of the NVL, the ascending afferent flow to the cortex seems to be reduced. This results in diminishing of the cortical neurones tone and readjusts the descending influences upon the NVL neurones activity.     

Differential effect of pentobarbital on chick neurons and astrocytes grown in culture

Primary cultures of neurons and astrocytes prepared from brains of 8-day-old and 15-day-old chick embryos. respectively, were grown for periods between 3 and 23 days. Cellular oxygen consumption was measured at various times in the presence of either pyruvate or succinate as substrate. Neuronal oxygen consumption was significantly higher than glial respiration, irrespective of the substrate employed. Dose-response curves for the effect of pentobarbital on respiratory activity of each cell type were constructed with the two substrates. In the presence of succinate neuronal respiration was more sensitive to pentobarbital than that of glial cells with a shift in the dose-effect curve by at least one order of magnitude. In the presence of pyruvate, glial cell respiration was inhibited at pentobarbital concentrations more than ten times lower than those effective in neurons. It is concluded that the differential sensitivity to pentobarbital between neurons and glia is due to differences in their respective energy metabolism.     

The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain

This study has investigated the possibility that acetylcholinesterase could play a non-classical role as an adhesion factor or growth factor in the development of dopaminergic neurons in organotypic slice culture of postnatal day 1 rats. When the culture medium was supplemented with acetylcholinesterase (3 U/ml), outgrowth of tyrosine hydroxylase-immunoreactive neurites was significantly enhanced. Addition of a specific inhibitor of acetylcholinesterase, BW284c51, caused a decrease in the number of tyrosine hydroxylase neurons and a reduction in the cell body size and extent of neurite outgrowth of remaining neurons. However, echothiophate which also inhibits AChE activity, did not produce these effects. Therefore acetylcholinesterase could act as a growth enhancing factor for dopaminergic neurons, and disruption of an as yet unidentified site on the acetylcholinesterase molecule by BW284c51 could decrease the survival and outgrowth of these neurons.