Biomedical subjects

P G Clarke

Publications and source records attributed to P G Clarke.

At least 19 recordsLinked to original sources

PubMed2000

Response of macrophage/microglial cells to experimental neuronal degeneration in the avian isthmo-optic nucleus during development.

Blockade of the retrograde axonal transport of isthmo-optic nucleus (ION) neurons in the avian embryo results in their massive degeneration. We used this system to investigate the response of macrophage/microglial cells to neuronal degeneration in the embryonic brain. Colchicine was injected into the right eye of quail or chick embryos at a time when the survival of ION neurons depends on retrograde trophic support from the retina, and the chronology of the subsequent macrophage/microglial response in the ION was analyzed. This response was restricted to the ION contralateral to the injected eye; no modifications of the normal state were observed in the surrounding parenchyma or in the opposite ION, used as control. The response was first detected 18 hours after the colchicine injection (18 hours pi), when an increase of the macrophage/microglial cell number was evident. The number of these cells in the affected ION increased, peaking at 40-48 hours pi. At later survival times, macrophage/microglial cells were progressively less abundant in the affected ION, which gradually diminished in size. At 120 hours pi the only remnant of the ION was a small cluster of macrophage/microglial cells, surrounded by a clear area with scarce nonmicroglial cells, in the region formerly occupied by the ION. This study reveals that a strong macrophage/microglial response occurs in the embryonic brain in response to neuronal degeneration but that these cells do not trigger the neuronal death, as they only appear after pyknotic fragments are already observable.

Animals↗

PubMed2000

Neuroprotective effects of a new glutathione peroxidase mimetic on neurons of the chick embryo's retina.

During their period of naturally occurring neuronal death, retinal ganglion cells are particularly vulnerable to axotomy. The resulting cell death requires protein synthesis and is redox-regulated, since antioxidants protect axotomized-ganglion cells when given in doses that maintain the redox status near an optimal set-point. Here we report the effects of BXT-51072, a new glutathione peroxidase mimetic, on ganglion cell death induced in various ways in the retinas of chick embryos. The intraocular injection of BXT-51072 protected axotomized neurons at doses in a narrow (tenfold) range. It also reduced the deleterious effects of intraocular tert-butyl hydroperoxide, an inducer of lipid peroxidation, and diminished the excitotoxic degeneration induced by N-methyl-D-aspartate. However, BXT-51072 did not noticeably reduce naturally occurring cell death. Globally, our results show that BXT-51072 has numerous protective effects in the retina. In accordance with published data, the present report indicates that glutathione peroxidase mimetics may have potential applications for neurologic or degenerative diseases.

Animals↗

PubMed1999

Inhibitors of mitogen-activated protein kinases protect axotomized developing neurons.

Axotomy kills developing neurons by mechanisms dependent on protein synthesis and influenced by the redox status. Amongst the redox-regulated transduction systems regulating gene expression are the mitogen-activated protein kinases (MAPKs). In the chick embryo, inhibitors of two different MAPK pathways, including notably the p38 kinase pathway, reduce the number of dying axotomized retinal ganglion cells. The regulation of the genetic events associated to axotomy-induced death thus seems to involve MAPKs.

Animals↗

PubMed1999

Protection of axotomized ganglion cells by salicylic acid.

Neuronal survival is influenced by the redox environment, and it has been shown that antioxidants protect developing neurons from the effects of axotomy. Here, we show that the intraocular injection of salicylic acid (SA) reduces the number of dying axotomized ganglion cells in the chick embryo. The antioxidant properties of SA are probably responsible for its protective effects, whose U-shaped dose-dependency matches that of several other antioxidants. We conclude that SA protects axotomized neurons by maintaining the redox status near an optimal set-point.

Animals↗

PubMed1999

Role of nitric oxide in a fast retrograde signal during development.

Two retrograde signals influence the chick embryo's isthmo-optic nucleus, which projects to the retina: a slow-acting survival signal due to uptake of neurotrophic factors, and a fast-acting death signal initiated by calcium entry into isthmo-optic terminals due to electrical activity. The latter signal also affects dendritic reorganization. Since nitric oxide synthase is present in isthmo-optic terminals and their retinal target cells, we have tested its possible role in the fast-acting signal. Intraocular injection of nitric oxide antagonists led within 6-12 h to a reduction in the number of dying isthmo-optic neurons and slowed dendritic reorganization. Surprisingly, nitric oxide agonists had a similar fast effect on neuronal death. Although the mechanism appears to be complex, nitric oxide is involved in mediating the fast-acting retrograde signal.

Animals↗

PubMed1999

The role of neuronal death during the development of topographically ordered projections: a computational approach.

At the time of synaptogenesis typically 50% of the neurons die. The biological role of this is still unclear, but there is evidence in the visual system that many neurons projecting to topographically inappropriate parts of their target are eliminated to improve the accuracy of the mapping. The signaling that determines neuronal survival involves electrical activity and trophic factors. Based on these observations, we have elaborated a computational model for the self-organization of a two-layered neural network. We observe changes in the topographical organization between the two layers. In layer 1, a traveling wave of electrical activity is used as input. Activity transmission to layer 2 can generate, according to a Hebbian rule, a retrograde death signal that is compensated by a trophic survival signal generated by the target cells. Approximately 50% of the neurons die, and we observe refinement in the topography between the two layers. In alternative versions of the model, we show that an equivalent reorganization can occur through Hebbian synaptic modification alone, but with less precision and efficiency. When the two mechanisms are combined, synaptic modification provides no further improvement over that produced by neuronal death alone. This computational study supports the hypothesis that neuronal death during development can play a role in the refinement of topographical projections in the nervous system.

Animals↗

PubMed1999

Relationships between neuronal death and the cellular redox status. Focus on the developing nervous system.

During the development of the nervous system, a large number of neurons are eliminated through naturally occurring neuronal death. Many morphological and biochemical properties of such dying neurons are reminiscent of apoptosis, a type of death involving the action of genetically-programmed events but also epigenetic phenomena including oxidative stress. The following review contains three parts focusing respectively on basic knowledge of neuronal death and redox regulation, the mechanisms involved in neuronal death which are ordered in three sequential phases, and on the complex relations between neuronal fate and the redox status. Finally, we point out that oxidants are not always detrimental for neuronal survival. On the one hand, dying neurons often display signs of oxidative stress, including an elevation of their intracellular concentration of free radicals. Antioxidants may reduce the extent of neuronal death, suggesting a causal implication of free radicals in the death-process. On the other hand, at high concentrations antioxidants may lose their protective effects on developing neurons, and a non-lethal oxidative stress may potentiate the protective effects of other agents. These data suggest that free radicals, perhaps through their effects on cellular signalling pathways, may have positive effects on neuronal survival, provided that their intraneuronal concentrations are maintained at low levels. Much evidence suggests that the neuronal redox status must be maintained within a narrow range of values compatible with survival. Antioxidants may protect neurons subjected to an oxidative stress following axotomy or trophic factor-deprivation; but excessive reduction may become equally detrimental for neurons.

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PubMed1999

Fast retrograde effects on neuronal death and dendritic organization in development: the role of calcium influx.

Retrograde signals from axon terminal to cell body are known to regulate neuronal survival and differentiation during development. They are generally attributed to the uptake and transport of trophic factors, but there is recent evidence in the isthmo-optic nucleus for a remarkably fast-acting retrograde signal from the contralateral retina that is not mediated by the conventional trophic route. The isthmo-optic nucleus undergoes 55% neuron death between embryonic days 12 and 17, and becomes laminated at embryonic day 14 owing to dendritic re-organization. Blockade of retinal electrical activity just before day 14 reduces neuronal death and lamination in the isthmo-optic nucleus within as little as 6 h. We here investigate how action potentials initiate the fast-acting retrograde signal, and we provide evidence that the first step is calcium entry into the isthmo-optic axon terminals. Neuronal death and lamination are rapidly reduced in the isthmo-optic nucleus by intraocularly injected omega-conotoxin, a blocker of N-type calcium channels known to be located mainly on axon terminal. Similar effects occurred with two other calcium channel blockers (cadmium and alpha-bungarotoxin) believed to act on both the isthmo-optic terminals and their target cells, but not with nifedipine, a blocker of L-type (mainly somatic) channels, supporting a presynaptic initiation of the fast signal. Nevertheless postsynaptic events may also be involved because pharmacological destruction of the amacrine targets cells of the isthmo-optic nucleus reduced its cell death and lamination 9-12 h later.

Animals↗

PubMed1999

An optimal redox status for the survival of axotomized ganglion cells in the developing retina.

The neuronal redox status influences the expression of genes involved in neuronal survival. We previously showed that antioxidants may reduce the number of dying ganglion cells following axotomy in chick embryos. In the present study, we show that various antioxidants, including the new spin trap azulenyl nitrone and 1,3-dimethyl-2-thiourea, protect axotomized ganglion cells, confirming that neuronal death involves an imbalance of the cellular redox status towards oxidation. However, high concentrations of antioxidants did not protect ganglion cells, suggesting that excessive reduction is detrimental for neurons. Simultaneous injections of two different antioxidants gave results only partly supporting this view. Combinations of azulenyl nitrone and N-acetyl cysteine in fact gave greater protection than either antioxidant alone, whereas N-acetyl cysteine lost its neuroprotective effects and diminished those of alpha-phenyl-N-tert-butyl nitrone when the two compounds were injected simultaneously. The results of the combined treatments suggest that azulenyl nitrone and alpha-phenyl-N-tert-butyl nitrone do not have the same chemical effects within the ganglion cells. Moreover, N-acetyl cysteine's own antioxidant properties enhance the spin trapping effects of azulenyl nitrone but potentiate the toxicity of alpha-phenyl-N-tert-butyl nitrone. Our main conclusion is that neuronal survival requires the maintenance of the redox status near an optimal set-point. "Reductive stress" may be as dangerous as oxidative stress.

Acetylcysteine↗

PubMed1998

Cooperation between glutathione depletion and protein synthesis inhibition against naturally occurring neuronal death.

It is generally agreed that naturally-occurring neuronal death in developing animals is dependent on the synthesis of proteins. Oxidative stress, as when intracellular concentrations of free radicals are raised or when cell constituents such as membrane lipids or protein thiols are oxidized, is also involved in various types of neuronal death. In the present report, we show that the number of naturally dying retinal cells in the chick embryo can be reduced by intraocular injections of cycloheximide, an inhibitor of protein synthesis. L-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthesis, can either enhance or diminish the cell death, depending on the conditions of treatment. Moreover, when the two inhibitors are combined, L-buthionine-[S,R]-sulfoximine potentiates the neuroprotective effects of cycloheximide. Measurements of retinal glutathione concentration and protein synthesis show the specificity of the treatments: buthionine-sulfoximine diminishes glutathione concentrations but not protein synthesis whereas cycloheximide inhibits protein synthesis without decreasing glutathione concentrations. Naturally-occurring neuronal death thus seems to involve the synthesis of proteins, and is also influenced by oxidative phenomena. Our results extend previous data in tectal-lesioned embryos, and suggest that a moderate, non-lethal oxidative stress can enhance the resistance of ganglion cells that might otherwise have died (spontaneously or following axotomy) owing to insufficient retrograde trophic support.

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PubMed1998

Neuronal death in the central nervous system during development.

About half the neurons in the brain die at the time when their connections are being formed. This neuronal death is regulated by anterograde and retrograde signals that reflect both electrical activity and the uptake of trophic factors. Our recent data on the isthmo-optic projection indicate that there are in fact two different retrograde signals: a slow-acting survival signal mediated by a neurotrophin, and a fast-acting death signal mediated by calcium entry due to electrical activity in the presynaptic terminals. The developmental roles of the cell death are not well understood, but they appear to include the elimination of aberrant connections. The intracellular mechanisms of the cell death may not always correspond to the apoptotic ones so thoroughly investigated in vitro, because only one of the three morphological types occurring regularly in vivo resembles apoptosis. However, our experiments on retinal ganglion cells indicate that several apoptotic mechanisms apply in this particular in vivo situation: these include an involvement of oxygenated free radicals and glutathione, cell cycle-related events, and probably the synthesis of proteins promoting neuroprotection or cell death.

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PubMed1997

Inhibition of glutathione synthesis can enhance cycloheximide-induced protection of developing neurons against axotomy.

Developing neurons depend for survival on target-derived trophic substances. These are thought to block the expression of a genetic program of cell death. Nevertheless, it is known that less orderly events such as oxidative stress are involved in neuron death. In vivo, retinal ganglion cell death induced by axotomy can be reduced by antioxidants. In this study, we investigated the effects of inhibiting glutathione synthesis by means of buthionine sulfoximine to characterize the influence of endogenous glutathione-dependent antioxidant systems on ganglion cell death. Moreover, since protein synthesis inhibition by cycloheximide has been shown to enhance glutathione synthesis in vitro, we studied the effects on cell death of intraocular injections of buthionine sulfoximine, cycloheximide and combinations of the two inhibitors. Cycloheximide's protective action did not seem to involve an increase in glutathione synthesis. Surprisingly, buthionine sulfoximine injected before cycloheximide enhanced its protective effects, whereas it inhibited them when injected later. We interpret our results as an interaction between death-promoting effects of glutathione depletion through an elevation of free radical concentrations and cycloheximide-sensitive effects of oxidative stress through the synthesis of both death-inhibiting and death-promoting proteins.

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PubMed1997

Presynaptic initiation by action potentials of retrograde signals in developing neurons.

Until recently, the only means by which electrical activity was believed to initiate retrograde signals was via postsynaptic events: modulated synthesis or release of trophic factors. We have evidence in chick embryos for a presynaptic initiation of retrograde signals from the retina to the isthmo-optic nucleus, which is known to undergo 55% neuron death between embryonic days 12 and 17 and to become laminated during this period. Intraocular injections of saxitoxin just before embryonic day 14 reduce neuron death and prevent lamination in the isthmo-optic nucleus within as few as 6 hr. We show that these rapid effects are attributable to the direct action of saxitoxin on the isthmo-optic terminals. Alternative possibilities, such as an indirect effect via the target cells, are ruled out by control experiments. Normally, action potentials may lead to a chain of second messenger events in the axon terminal that is signaled retrogradely via the transport of a long-lived second messenger.

Action Potentials↗

PubMed1997

Early retrograde effects of blocking axoplasmic transport in the axons of developing neurons.

Depriving developing neurons of retrograde trophic support may disrupt their development and often causes them to die. We here report the effects, in chick embryos, of eliminating retrograde support in the isthmo-optic projection by blocking axoplasmic transport in the terminal parts of the axons, which is known ultimately to kill the isthmo-optic neurons. Within only 9 h, this had perturbed the process of cellular reorganisation that eventually leads to the laminated appearance of the mature isthmo-optic nucleus. Neuron survival in the isthmo-optic nucleus was affected even more quickly, but the earliest change, occurring in as little as 3 h, was not an increase in the number of dying neurons, but a decrease below control values. This novel effect was still present at 6 and 9 h after the injection, but at longer survival times the number of dying neurons increased well above control values as expected. Our interpretation of the transient decrease in neuronal death is that retrograde trophic signals include both death-promoting and life-promoting components, and that the former act faster in this system.

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PubMed1996

Axotomy-induced retinal ganglion cell death in development: its time-course and its diminution by antioxidants.

Developing neurons die when deprived of trophic support from their axonal target. Although this is generally attributed to the programmed expression of suicide proteins, recent data suggest that a less orderly mechanism involving oxidative stress may also be involved. We have studied retinal ganglion cell death in the chick embryo after a contralateral tectal lesion. The kinetics of cell death, as judged from counts of pyknotic cells, are described. In addition, we show that the pyknotic counts are reduced following intraocular injections of the protein synthesis inhibitor cycloheximide or the antioxidants N-t-butyl-alpha-phenylnitrone and N-acetyl cysteine. Our results suggest that target deprivation-induced ganglion cell death involves oxidative stress.

Acetylcysteine↗

PubMed1996

Competitive exclusion between axons dependent on a single trophic substance: a mathematical analysis.

A mathematical model is presented of competition between axons for a trophic substance, such as is believed to occur particularly during development. The model is biologically realistic. The growth-stimulating activity of the trophic molecules is assumed to result from their binding to high-affinity receptors on neurons and their axons, but the model also incorporates uptake by nonneuronal cells possessing only lower affinity receptors. Plausible and fairly general assumptions are made concerning the kinetics of binding and internalization and the effects on axonal growth. The model takes into account the possibility that trophic factor production may be regulated by the afferent axons or autoregulated. The variables specified are the "axonal vigor" of each axon, representing the ability of each axon to take up trophic molecules, and the concentration of trophic molecules in the extracellular space of the axonal target region. Of the several parameters introduced, the most important turns out to be the "zero vigor-growth parameter," which is defined as the concentration of trophic molecules that gives zero growth of the vigor of a given axon. By means of a Lyapunov function, it is shown that the system will approach asymptotically to a stable equilibrium characterized by the survival of only the axon whose zero-growth parameter is lowest. Or, if several axons share the same lowest zero-growth parameter, these will all survive. The model may be particularly relevant to the elimination of polyneuronal innervation from developing muscle fibers and from autonomic ganglion cells.

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PubMed1996

Retrograde neurotrophin-mediated control of neurone survival in the developing central nervous system.

Neuronal death in vertebrate development is widely believed to be regulated by retrograde survival signals from the axonal target territory, and these signals are assumed to be initiated by the binding of trophic molecules to the axon terminal. However, direct evidence for the retrograde transmission of such survival signals along developing axons is only available in the peripheral nervous system. We show here in a central projection, the isthmo-optic projection of chick embryos, that a neurotrophin, brain-derived neurotrophic factor, can indeed initiate retrograde survival signals from the target territory. A related molecule, neurotrophin-3, is ineffective.

Animals↗

PubMed1996

Dendritic reorientation and cytolamination during the development of the isthmo-optic nucleus in chick embryos.

In the mature isthmo-optic nucleus (ION, source of efferents to the contralateral retina), the neuronal perikarya are generally described as being arranged in a single convoluted lamina surrounding a U-shaped region of neuropil, into which their highly polarized (unidirectional) dendritic arbors project perpendicularly. We find, however, that the details are more complicated than this description suggests, and are variable, as might be expected if the ION is self-organized through neuron-to-neuron interactions in development. The laminated conformation of the ION first appears at embryonic day (E) 14. Our previous experiments indicate that this involves the displacement of perikarya and is not due to sculpting by neuronal death. We here present a quantitative demonstration that the dendritic arbors reorient during the period of lamination. At E11, they are already highly polarized, but their directions are different from those in the adult, being mostly medio-rostro-ventral. Then, between E11 and E13, the arbors in the border region of the ION undergo major changes in their direction of polarization, projecting towards the center of the ION. The arbors within the core of the ION make more subtle changes. The dendritic reorganization seems to be intrinsically linked to the process of cytolamination, since the two events occur synchronously and disruption of either affects the other. Mechanisms are discussed; interaction with afferents is not responsible for lamination.

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