Inactivation of the glial fibrillary acidic protein gene, but not that of vimentin, improves neuronal survival and neurite growth by modifying adhesion molecule expression.

Intermediate filaments (IFs) are a major component of the cytoskeleton in astrocytes. Their role is far from being completely understood. Immature astrocytes play a major role in neuronal migration and neuritogenesis, and their IFs are mainly composed of vimentin. In mature differentiated astrocytes, vimentin is replaced by the IF protein glial fibrillary acidic protein (GFAP). In response to injury of the CNS in the adult, astrocytes become reactive, upregulate the expression of GFAP, and reexpress vimentin. These modifications contribute to the formation of a glial scar that is obstructive to axonal regeneration. Nevertheless, astrocytes in vitro are considered to be the ideal substratum for the growth of embryonic CNS axons. In the present study, we have examined the potential role of these two major IF proteins in both neuronal survival and neurite growth. For this purpose, we cocultured wild-type neurons on astrocytes from three types of knock-out (KO) mice for GFAP or/and vimentin in a neuron-astrocyte coculture model. We show that the double KO astrocytes present many features of immaturity and greatly improve survival and neurite growth of cocultured neurons by increasing cell-cell contact and secreting diffusible factors. Moreover, our data suggest that the absence of vimentin is not a key element in the permissivity of the mutant astrocytes. Finally, we show that only the absence of GFAP is associated with an increased expression of some extracellular matrix and adhesion molecules. To conclude, our results suggest that GFAP expression is able to modulate key biochemical properties of astrocytes that are implicated in their permissivity.

Synaptic and non-synaptic AMPA receptors permeable to calcium.

For a long time, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors permeable to calcium have been considered to be either non-existent or as "atypical". There is now ample evidence that these receptors exist in numerous regions of the nervous system and in many neuronal as well as non-neuronal cell populations. This evidence has been accumulated by several methods, including electrophysiological recording, calcium imaging and cobalt-loading. Functional AMPA receptors permeable to calcium are already expressed at very early stages of embryonic development, well before the onset of synaptogenesis. They are probably involved in the paracrine signaling necessary for construction of the nervous system before becoming involved in synaptic transmission. In immature cells, cyclothiazide strongly increases the steady-state level of responses not only to AMPA, but also to kainate. Ingestion, during pregnancy, of food or drug substances that can cross the placental barrier and act upon the embryonic receptors may constitute a risk for normal development. In the adult nervous system, synaptic as well as non-synaptic (paracrine) AMPA receptors permeable to calcium are probably widely expressed in both glial and neuronal cells. They may also participate in controlling some aspects related to adult neurogenesis, in particular the migration of newly formed neurons.

Blockade of AMPA/kainate receptors can either decrease or increase the survival of cultured neocortical cells depending on the stage of maturation.

Neurotoxicity has often been associated with glutamate receptor stimulation and neuroprotection with glutamate receptor blockade. However, the relationship may be much more complex. We dissociated cells from the rat neocortical anlage at an early stage of prenatal development (embryonic day 14). The cells were exposed in vitro to agonists and antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate and N-methyl-D-aspartate (NMDA) receptors and the effects on differentiation and survival have been quantitatively and qualitatively evaluated. NMDA and the non-competitive antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801) had the expected effects (the agonist decreasing and the antagonist increasing neuronal survival) when applied at a relatively advanced stage of in vitro maturation, but no significant effect in either direction at earlier stages. Kainate also had an effect on cell survival only at an advanced stage (where it decreased the number of cells). However, this cannot be attributed to the absence of functional AMPA/kainate receptors at earlier stages, since: (1) cells could be loaded with cobalt; and (2) early application of kainate dramatically reduced the number of cobalt-positive cells. Furthermore, exposure at early stages to 6,7-dinitroquinoxaline-2,3-dione (DNQX), or GYKI 53655, (competitive and non-competitive AMPA receptor antagonists, respectively) strongly reduced cell survival. The effects were concentration- and time-dependent with a complex time--curve. The decrease in cell number was maximal after antagonist application from 2 to 5 days in vitro. The effects of DNQX could be cancelled by co-application of kainate. When exposed to an antagonist at later stages of development, the number of surviving cells gradually approached control values and finally became significantly higher. Our results suggest that cells of the developing neocortex (and perhaps newly generated cells in the adult brain) require at different stages of their development, an appropriate level of AMPA/kainate receptor activation.

AMPA receptor activation leads to neurite retraction in tangentially migrating neurons in the intermediate zone of the embryonic rat neocortex.

In rat (König et al. [1998] 28th Annual Meeting of the Society of Neuroscience, Los Angeles. 24:314.6) and mouse (Métin et al. [2000] J. Neurosci. 20:696-708), neurons migrating tangentially in the intermediate zone (IZ) of the neocortical anlage express functional AMPA receptors permeable to calcium. The role of these receptors is as yet unknown. We exposed organotypic cultures of rat telencephalon (embryonic day 15) to AMPA receptor agonists or antagonists, and analyzed the effects of these treatments on cells in the IZ labeled with antibodies against the isoforms a, b and c of microtubule associated protein 2 (MAP2) and the polysialylated neural cell adhesion molecule (PSA-NCAM). The presence of functional AMPA receptors permeable to calcium was checked by cobalt-loading. After exposure to AMPA alone for at least 6 hr, we observed a significant increase in the number of rounded, MAP2 positive cells in the IZ close to the migratory front. When AMPA was combined with cyclothiazide, the increase was already significant after 3 hr. These effects were dose-dependent and could be partially or totally blocked by DNQX or GYKI 53655 respectively, that suggests that they are mediated by AMPA receptors. Paracrine AMPA receptor activation might participate, together with other signals, in guiding the migratory stream, or provide stop signals for migrating cells.

In vitro neuroprotection against glutamate toxicity provided by novel non-competitive N-methyl-D-aspartate antagonists.

Primary cultures of embryonic cortical cells were used to test the neuroprotective effect of two novel non-competitive N-methyl-D-aspartate antagonists against the neurotoxicity of glutamate. These two compounds, GK 11 and GK 222, are phencyclidine derivatives that contain asymetric carbon atoms. We have tested the neuroprotective effects of the racemates as well as those of the corresponding plus and minus enantiomers and have compared them with the reference drug dizocilpine (MK-801). We show here that the (-) enantiomers have a significantly better protective effect than the (+) enantiomers, but that the former are as efficient as the corresponding racemates. Qualitative analysis with different neuronal and glial markers does not reveal any difference in the population of protected cells among the different molecules tested.

AMPA/kainate receptors modulate the survival in vitro of embryonic brainstem cells.

This study aimed at analyzing the involvement of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) receptors in the survival of cultured rat embryonic brainstem cells, dissociated on embryonic day 14. The cell number was estimated after pharmacological manipulation of the receptors by exposure to agonists or antagonists. The developmental stage at the moment of drug application was critical for cell survival. We observed after 8 days in vitro a much stronger decrease in the number of gamma-enolase-positive cells when the cultures were treated for 3 days with the antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) starting on the day of plating than when DNQX was added after 5 days in vitro. Conversely, exposure to the agonists (RS)-2-amino-3-(3-hydroxy-5-tri-fluoromethyl-4-isoxazolyl)-propion ic acid (T-AMPA) or kainate for 3 days significantly reduced cell survival only when the treatment was initiated after 5 days in vitro. Survival of S-100-positive cells was not affected after exposure to either agonists or antagonists. Neither agonist nor antagonist treatment modified cell proliferation, as assessed by 5-bromo-2'-deoxyuridine (BrdU) staining, suggesting that the decrease in the number of gamma-enolase-positive cells is essentially due to cell death. If some of the processes we observed in vitro correspond to analogous events in vivo, then exposure to excitatory amino acid receptor agonists or antagonists at critical stages of embryogenesis may alter the development of the central nervous system.

Immunocytochemical study of phenotypic plasticity of cultured dorsal root ganglion neurons during development.

Rat dorsal root ganglia (DRG) were cultured from different stages of development ranging from embryonic day-14 to adult. The expression of eight neurotransmitter phenotypes was examined with immunocytochemical detection and the percentages of each phenotype were calculated with reference to the whole neuronal population defined by the expression of neuron-specific enolase (NSE). The expression of peptides, calcitonin gene-related peptide (CGRP), substance P (SP), cholecystokinin (CCK) and neuropeptide Y (NPY) was always present whatever the age at onset of the cultures. Although the percentage of CGRP remained stable, that of the other peptides declined progressively. Their in-vitro expression did not differ markedly from that found in vivo. Another group of neurotransmitters, including 5-hydroxytryptamine (5-HT), thyrotropin-releasing hormone (TRH) and gamma-aminobutyric acid (GABA) was never expressed in situ in DRG neurons. In culture, they were expressed in a high percentage of neurons, especially for 5-HT and TRH, and they showed a similar evolution, with a decrease at early postnatal ages followed by a further increase. This profile suggests that the expression of these transmitters is strongly environment-dependent and may be repressed in situ. Finally, somatostatin (SOM) was found only in cultures prepared from adult tissues, whereas it was present in situ from the embryo onwards. The expression of this peptide would thus require a stabilization by a long exposure to environmental factors. We can conclude that the great diversity of phenotypic expression found in DRG neurons in situ is the result of a wide variety of influences occurring at different stages of development in a large potential repertory present in these neurons.

Fictive motor activities in adult chronic spinal rats transplanted with embryonic brainstem neurons.

The present study was designed to examine the effects of an intraspinal transplantation of embryonic brainstem neurons on fictive motor patterns which can develop in hindlimb nerves of adult chronic spinal rats. Seventeen adult rats were spinalized at T8-9 level and, 8 days later, a suspension of embryonic cells obtained either from the raphe region (RR, n = 8) or from the locus coeruleus (LC, n = 9) was injected caudally (T12-13) to the cord transection. Eight control animals (control rats) were spinalized and injected with vehicle under the same conditions. One to three months later, the animals were decorticated and fictive motor patterns were recorded in representative hindlimb nerves. The data revealed that both control and grafted spinal rats could exhibit two distinctly different fictive motor patterns, one which could be associated with stepping and the other with hindlimb paw shaking. They further showed that following transplantation of embryonic RR or LC neurons the excitability of the spinal stepping generator was increased, whereas that of the spinal neural circuits which generate hindlimb paw shaking was not significantly affected. A histological analysis performed on the spinal cord segments below the transection revealed complete absence of serotonin and noradrenaline immunoreactivity in control spinal animals and, in both types of grafted rats, an extensive monoaminergic reinnervation with synaptic contacts between monoaminergic transplanted neurons and host interneurons and/or motoneurons. The possible mechanisms by which grafted monoaminergic neurons can influence the spinal motor networks are discussed.

AMPA elicits long-lasting, partly hypothermia-sensitive calcium responses in acutely dissociated or cultured embryonic brainstem cells.

This study aimed at testing if, and under which conditions, long-lasting cytosolic calcium responses can be induced in dissociated embryonic brain cells exposed to alpha-amino-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor agonists. Rat brainstem cells (gestation days 13-14; mean crown-rump lengths 8-11 mm) were mechanically dissociated and loaded with the fluorescent calcium marker Fluo-3 after in vitro delays ranging from 20 min to 6 days. The cells were exposed to various concentrations of AMPA, domoic acid or kainic acid. The evoked fluorescence changes, indicating variations of cytosolic calcium, were recorded and analysed either with a video-microscope or a laser cytometer. Even at the earliest stages, non-desensitizing (or partly desensitizing) calcium responses to AMPA were found. In addition, sequential exposure to AMPA followed either by domoic acid, or by AMPA in the presence of aniracetam, revealed the existence of cells bearing predominantly desensitizing receptors. The non-desensitizing as well as desensitizing response components were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX). When the experiments were conducted at 24 degrees C, the cytosolic calcium levels generally returned close to pre-stimulus baseline levels after washout. In contrast, when the working temperature was slightly raised (to 27 degrees C), complex secondary calcium rises were observed not only during prolonged stimulation, but also after short agonist application. The calcium modulation might be correlated with some form of cellular "learning" in the embryonic brain. Under particular conditions, where the regulation processes are either switched off by cell programmes or simply overloaded, the cascade of events comprising secondary calcium rises may lead to cell death.

Protection by BTCP of cultured dopaminergic neurons exposed to neurotoxins.

This study presents 'in vitro' evidence for a protection of cultured dopaminergic neurons against the toxicity of 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+) by pretreatment with BTCP, a selective and potent dopamine (DA) uptake blocker. Moreover, we show that, at low concentration (10 nM), treatment with MPP+, which is more selective than 6-OHDA for dopaminergic neurons, is followed by some regeneration of these neurons.

Early functional glutamate receptors in acutely dissociated embryonic raphe cells.

We report for the first time, modulation of cytosolic calcium in response to glutamate and specific glutamate receptor agonists in early embryonic rat brain cells (raphe cells taken at gestation days 13 or 14). Metabotropic as well as ionotropic agonists were effective. Cells responding to kainic acid were particularly prominent in caudal raphe. We used very short post-plating delays (2 to 6 h); it may therefore be assumed that functional receptors already exist in the intact embryonic brain by gestation day 13. Since many developmental processes are influenced by cytosolic calcium modulation, glutamate receptors may play a key role in brain development, well before the extensively studied postnatal peak in receptor density.

Networks formed by dorsal root ganglion neurites within spinal cord explants: a computer-aided analysis of HRP intracellularly labeled neurons.

Dorsal root ganglion (DRG) neurons from rat embryos were explanted either alone or with the attached spinal cord (SC). Neuritic processes were mapped out histologically using an intracellular iontophoretic HRP method. Computer reconstructions and morphometric parameters allowed a comparative quantitative analysis of the DRG neurons and of their neuritic processes in both models. The first model, strongly dependent on nerve growth factor (NGF) for its survival, developed large multidirectional processes. The second model showed a bipolar distribution of its neuritic processes, the central one entering predominantly the dorsolateral part of the cord explant and ramifying both homo- and heterolaterally. The quantitative data revealed a significant decrease in the overall size of the neuritic networks of the second model (with attached SC). A discriminant analysis permitted the recognition of these two populations of DRG neurons. The role of the spinal cord explant, and more precisely the target cells of the dorsal horn, was considered to be a prominent factor in the development of the DRG neuritic networks.

Fate of B1-B2 and B3 rhombencephalic cells transplanted into the transected spinal cord of adult rats: light and electron microscopic studies.

Embryonic cell suspensions (14-day embryos) containing either B3 or B1-B2 serotonergic cell groups were obtained by microdissection of specific rhombencephalic regions and transplanted into the transected spinal cord of adult male Sprague-Dawley rats. After 3 months of survival, the animals were sacrificed and the spinal cords processed for the immunocytochemical detection of serotonin (5-HT). 5-HT-immunoreactive fibers from B1-B2-grafted cells were selectively distributed in the ventral horn and the intermediolateral cell column (IML) where they established conventional synaptic contacts. However, B3 5-HT cells grew and extended their processes into the dorsal horn where in addition we observed scarce synaptic contacts as in the normal spinal cord. These results suggest that the specificity of the 5-HT innervation of the spinal cord by grafted neurons is due, at least partly, to the presence of local mechanisms mediating guidance and cell recognition, possibly operating in conjunction with preexisting substrate pathways.

Neuronal firing patterns of organotypic rat spinal cord cultures in normal and in ACTH/alpha-MSH(4-10) analog (BIM 22015)-supplemented medium.

The spontaneous and evoked electrical patterns of spinal cord explants from 13- to 14-day old rat fetuses grown from 2 to 8 weeks in vitro were compared when fed either with a standard or with an adrenocorticotropic hormone/alpha-melanocyte stimulating hormone (4-10) analog (BIM 22015)-supplemented medium. Standard and BIM 22015-treated cultures developed similar patterns of extracellularly recorded activity which consisted of mostly phasic but also tonic discharges. The standard cultures when treated by BIM 22015 in acute experiments (100 micrograms/ml) showed a decrease in their frequency of discharges which fired in a regular tonic pattern. These effects were neither age- nor dose-dependent but were increased in Ca2+ free medium. The ventral cord neurons chronically fed with BIM 22015 showed a strongly bursting pattern resembling strychnine-induced synchronized bursts. Both these effects, inhibitory (acute) and excitatory (chronic), of the BIM upon spinal cord cultured ventral horn neurons are discussed as possible calcium-dependent phenomena.

Transplantation of embryonic serotonin immunoreactive neurons into the transected spinal cord of adult monkey (Macaca fascicularis).

Five adult monkeys (Macaca fascicularis) underwent a total section of the spinal cord at the thoracic level (T6). Four of them received a daily treatment with cyclosporin (10 mg/kg). Ten days later, two animals treated with cyclosporin and one without cyclosporin received at T8 and T10 levels an injection of a cell suspension prepared from the rhombencephalon of a 40-day-old macaque embryo. Two control animals received one injection of Hank's balanced salt solution. The animals were sacrificed after 2 months (one grafted and one control) and 3 months (two grafted and one control), and their spinal cord was processed for the immunocytochemical detection of serotonin using light and electron microscopy. After 2 months of survival, serotonergic neurons had survived and developed within the transplant. Three months after transplantation, in the animal treated with cyclosporin, serotonergic neurons were found to survive with their axons growing into the host grey matter and establishing axosomatic and axodendritic synapses in the ventral horn. If the graft was isolated in the white matter no fibers were seen invading the grey matter.

Localization of dopamine carriers by BTCP, a dopamine uptake inhibitor, on nigral cells cultured in vitro.

BTCP, N-[1-(2-benzo(b)thiopenyl)cyclohexyl]piperidine, a derivative of phencyclidine, acted as a potent dopamine (DA) uptake blocking agent on primary cultures of dopaminergic neurons obtained from substantia nigra (IC50 = 70 nM). This value was closely related to IC50 determined for reference DA uptake inhibitors such as nomifensine (70 nM) or benztropine (50 nM), showing the specificity of BTCP towards the DA carrier. Thus, we used BTCP as a tool to visualize the DA uptake complexes on cultures, a model which preserves the integrity of the neurons. The [3H]BTCP binding sites directly visualized by radioautographical (RAG) labelling seemed to follow the fibres (axons or dendrites) of neurons in culture whereas the cell bodies were not labelled. The [3H]DA uptake visualized by RAG labelling, was inhibited either partially by BTCP at a concentration near its IC50 or totally by a high concentration of BTCP, all over the dopaminergic neurons (neurites and somas) immunostained with an anti-DA antiserum. Thus, the distribution of DA carriers can be investigated by a suitable tool, BTCP, a powerful and selective DA uptake blocker. These carriers have been visualized by radioautography with tritiated BTCP along the neurites, and the uptake can be totally blocked by a high concentration of BTCP all over DA neurons in vitro.

Non-competitive antagonists of N-methyl-D-aspartate prevent spontaneous neuronal death in primary cultures of embryonic rat cortex.

Primary cultures of embryonic rat cerebral cortex were treated after 17 days in vitro for 10 min with a single dose of the non-competitive antagonists of N-methyl-D-aspartate (NMDA) receptor MK 801, TCP, and GK 11. They were then maintained in vitro for 31, 59, or 73 days, and then processed for the immunocytochemical detection of neuron-specific enolase (NSE). Immunoreactive cells were counted in treated and control cultures, and it was found that, except at 31 days, treated cultures contained far more NSE immunoreactive cells than controls. Moreover, this effect was dose-dependent, since with both TCP and GK 11 neuron survival was significantly higher with, respectively, 20 microM and 5 microM than with the lowest concentration of 2.5 microM. We tentatively conclude that spontaneous neuron death occurring in primary cultures in vitro is at least partly related to the NMDA-associated Ca++ channel, since the common property of the molecules we used is to block this channel. The relevance of this mechanism of cell death in vitro to neuronal death in vivo is discussed.

Population-specific modulation of 5-HT expression in cultures of embryonic rat rhombencephalon.

This study aimed at analyzing the regulation of in vitro serotonin expression by neurons taken from different regions of the embryonic rat rhombencephalon. We studied the influence of co-culture with alarplate tissue using immunocytochemical and biochemical methods. Computer-assisted densitometry was used to estimate the co-culture effects on the serotonin content of the cell bodies. The more dynamic aspects of serotonin expression, such as synthesis and release, were studied by measuring (3H)serotonin newly synthesized from (3H)tryptophan. The density of the immunostaining was significantly decreased in B1,B2 cells by co-culture with both caudal and rostral alar-plate tissue. For B4-B9 cells, only co-culture with rostral alar-plate tissue produced a significant decrease. The de novo synthesis of serotonin was significantly decreased in B1,B2 neurons co-cultured with caudal alar-plate tissue only. Once again, the B4-B9 cells proved to be less influenced by the experimental conditions, as co-culture with both types of alar-plate tissue produced no significant effect. We concluded that the in vitro expression of serotonin can be modulated by environmental factors, but the relative influence of these factors is very different in rostral versus caudal serotonin expressing cell populations.

[First transplantation of embryonic serotonergic neurons in primate spinal cord]. / Première transplantation de neurones sérotonergiques embryonnaires dans la moelle épinière d'un primate.

Three adult monkeys (Macaca fascicularis) underwent a total section of the spinal cord at thoracic level (T6). 1 week later, two of them received at T8 level an injection of a cellular suspension prepared from the raphe region of a foetal macaque 39 days old. The third animal received one injection of buffer solution. 1 month later, the animals were sacrificed, and their spinal cord was processed for the immunocytochemical detection of serotonin with light and electron microscopy. Serotonergic neurons had survived after transplantation, and had grown axons and dendrites. Afferent and efferent synapses to serotonergic neurons were readily identified.

Influence of GM1 gangliosides on the growth of cultured rat embryonic serotonergic neurons.

GM1 gangliosides were added to the medium of cultured raphe neurons enriched in the serotonergic phenotype in order to study their influence on biochemical and morphological growth parameters of serotonergic neurons. After 2 days of culture in the presence of GM1, specific uptake of serotonin measured by scintillation counting exhibited a moderate but significant increase for a GM1 concentration of 5 X 10(-8) M. Morphological parameters of 5-HT neurons were measured after immunocytochemical staining with specific serotonin antiserum, and digitalization of immunoreactive cells. Eight parameters were studied; for concentrations of 5 X 10(-8) and 10(-7) M of GM1, the absolute neuritic field area and the total length of the segments were significantly increased, whereas the number of neuritic segments, and their mean length were not modified. We conclude that GM1 ganglioside has a significant influence on the growth of serotonergic neurons. Moreover, electron microscopy showed, on treated cultures, a dramatic increase of the number of spicules all along the neuron's process, suggesting that GM1 could act by modifying the attachment of cells to their substrate. The possible molecular mechanisms of the action of GM1 are discussed.