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Biomedical subjects

M C Calvet

Publications and source records attributed to M C Calvet.

At least 19 recordsLinked to original sources
PubMed1995

Cryopreserved neuronal cells in long-term cultures of dissociated rat cerebral cortex: survival and morphometric characteristics as revealed by immunocytochemistry.

Blocks of fresh tissue from embryonic rat cerebral cortex, dissociated either prior to or after freezing, were stored for up to 10 months in liquid nitrogen at -196 degrees C with 7% dimethylsulfoxide (DMSO) as cryoprotectant. Slow freezing and rapid thawing, a reduced medium volume and moderate elevation of both extracellular K+ (20 mM) and sera (20%) promoted survival of neurons (up to 7 weeks) on polylysine-coated glass coverslips. Although there was cell loss associated with freezing, the surviving cells developed morphological and immunocytochemical properties similar to those expressed by unfrozen cells when using anti-GFAP (glial fibrillary acidic protein), anti-NSE (neuron specific enolase) and anti-GABA (gamma-aminobutyric acid) antibodies. A comparative computer-aided analysis of the morphometric patterns of GABA-IR neurons allowed the individualization of two similar populations in both fresh and frozen controls. NSE- and GABA-immunoreactive (IR) cells were counted in frozen controls and thienyl-phencyclidine (TCP)-treated cultures. The latter yielded fewer cells whereas it was the opposite in fresh TCP-treated cultures. In view of these findings, a detailed analysis of the effects of both neuroprotective and neurotoxic agents on frozen cells is undertaken before considering that cryopreservation might be a suitable storage method for clinical trials of neuron grafting.

Animals↗
PubMed1994

Neuroprotective effect of N-methyl-D-aspartate non-competitive antagonists (arylcyclohexylamine derivatives) on human cultured spinal cord cells.

The non-competitive N-methyl-D-aspartate antagonist cis-Pip/Me 1-[1-(2-thienyl)-2-methyl-cyclohexyl] piperidine (GK11) either in racemic(+/-) or (-) enantiomeric form has been investigated on survival of human fetal spinal cord cells in culture. The treated cultured cells were processed for immunocytochemical detection of GABA at different time intervals ranging from 3 to 27 weeks. The number of GABA-stained cells was found to be greater in the treated cultures than in the control ones. The qualitative and quantitative morphological features of both control and treated cells were analysed by computer-assisted methods which allowed us to individualize three different populations: (1) all control and treated small neuritic field neurons, (2) young control and old treated large neuritic field neurons and (3) old control neurons with large neuritic fields.

Cells, Cultured↗
PubMed1993

Culture model for the study of cerebral malaria: antibodies from Plasmodium falciparum-infected comatose patients inhibit the dendritic development of Purkinje cells.

The in vitro development of kitten Purkinje cells was inhibited by sera or IgG from cerebral malaria (CM) patients but not by sera from acute non-complicated malaria (NCM) cases. This inhibitory effect, quantified by computer-assisted methods, concerned the dendritic (though not the axonal) development and was found to be related to the presence of CM sera of self-reactive antibodies of the IgG class. These results suggest that protection acquired against the major complication of Plasmodium falciparum malaria may correspond to the repression of an abnormal stimulation of autoreactive B cells.

Animals↗
PubMed1993

Immunocytochemical demonstration of monoamine-containing cells in human fetal brain stem dissociated cultures.

Primary dissociated cultures were prepared from the brain stems of 7- to 10-week-old human fetuses. Immunocytochemical detection of serotonin (5HT)-, dopamine (DA)-, noradrenaline (NA)- and tyrosine hydroxylase (TH)-containing cells was performed within 4 to 12 weeks in culture. We observed 5HT- and NA-stained neurons in the rhombencephalon. DA-stained cells were observed in the mesencephalon. TH-stained neurons were evidenced in both parts of the brain stem. Our in vitro system appears particularly appropriate for further investigation of grafts transplanted into the lesioned central nervous system (CNS).

Brain Stem↗
PubMed1992

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.

Animals↗
PubMed1992

TCP enhances the survival of human fetal spinal cord cells in culture.

Dissociated cell cultures were prepared from human spinal cords of 7-10-week-old fetuses. After 10 weeks progressive neuronal necrosis was observed in controls whereas N/1-(2-thienyl)cyclohexyl/piperidine (TCP) enhanced the survival time of the cells. After 21 weeks the number of gamma-aminobutyric acid (GABA)ergic and neuron specific enolase (NSE)-stained neurons was higher in the TCP-treated cultures than in controls. TCP appears to be a promising drug for long term survival of neurons.

Biomarkers↗
PubMed1992

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.

Adrenocorticotropic Hormone↗
PubMed1988

Computer-assisted analysis of the developing Purkinje neuron. I. Effects of the age of the animal at the moment of explantation on the subsequent dendritic development in organotypic cultures.

Purkinje cell dendritic arborization were studied in intracellularly horseradish peroxidase-stained mature neurons grown during 30-40 days in organotypic cerebellar cultures from kittens of various ages. The effects of the age of the animal at the moment of explantation upon the subsequent dendritic and axonal development were studied in kitten cerebella of 1, 12 and 21 postnatal days old. These effects were investigated by computer-assisted methods. Qualitative data were found to be different in these 3 in vitro models explanted at 3 different ages except for the spine development which remained the same. Quantitative data, obtained from 15 measurable parameters, showed that the degree of dendritic development was higher for the Purkinje cells explanted at the latest postnatal age: the older the animal, the larger the dendritic tree in culture. A discrimination analysis permitted a recognition of the 3 differently aged populations (94% well classified cells) based on 3 selected variables, number of primary dendritic trunks, number of rows and dendritic field areas. These results lend further support to the major role played in the final dendritic arrangement by extrinsic (discriminant factors, their pre-existence in vivo being responsible for the subsequent degree of the in vitro development of the Purkinje cell dendritic arbor.

Aging↗
PubMed1988

Computer-assisted analysis of the developing Purkinje neuron. II. A comparative study of the dendritic development in differently aged kittens and in organotypic cultures explanted at the same ages.

Golgi-prepared cerebella from 10-13-(model K) and 22-day-old (model C) kittens were analyzed and compared with 30-40 days in vitro (DIV) horseradish peroxidase (HRP)-stained organotypic cultures from kitten cerebella explanted at comparable ages (12 (model E) and 21 (model T) postnatal days). In addition, HRP-stained cerebella explanted from 1-day-old kittens were used as a reference (model N). Computer reconstructions and morphometric parameters (14 variables) allowed a comparative quantitative analysis of the Purkinje cell dendritic trees and axonal processes. The biplanar arrangement of the dendritic sheets observed in vivo at 10-13 days (K) was replaced by a mostly bipolar one in the in vitro Purkinje cells (E) explanted at the same age with their axon following the direction of either dendrite. The dendritic expansion observed in intact animals between 12 and 22 days was mostly due to an increase in the number of dendritic segments having shorter lengths rather than an increase in the number of rows. A discriminant analysis permitting the recognition of 3 populations of Purkinje cells in both groups reinforced the previous hypothesis that the dendritic shapes are modelled by specific afferents pre-existent in vivo.

Aging↗
PubMed1987

[Electrophysiology and analysis of the image of the development of Purkinje cells in culture and in the intact animal].

The electrophysiological and morphological properties of the Purkinje cells (P cells) grown in organotypic cultures were studied in HRP intracellulary labelled neurons and compared to Golgi stained P cells from kitten of different ages. The effects of afferent fiber depletions on the final dendritic topological parameters were investigated by 3-dimensional reconstructions and computerized methods. In culture, the P cell dendritic trees always appeared reduced in size when compared to their in vivo counterparts. A discriminant analysis permitted the recognition of 3 populations of cultured P cells according to the type of deafferentation. In intact animals, the dendritic organization appeared biplanar around day 13. These results demonstrate that the presence of all normal inputs is required to achieve the full elaboration and the monoplanar disposition of the P cell dendrites.

Aging↗
PubMed1985

Morphologic and functional abnormalities that develop in kitten Purkinje neurons during maintenance for months after maturation in organotypic cultures.

The morphologic and functional properties of the Purkinje cells (P-cells) grown for 10-11 weeks in organotypic cultures from newborn kitten cerebella were studied and compared to cultures which had been grown for 4-5 weeks under the same standard conditions. Electrophysiological and morphological data were obtained from HRP iontophoretically labeled neurons and were quantified by means of computerized techniques. Extracellular recordings of spontaneous activity showed that the 10-11-week-old P-cells had a pacemaker-like firing rate whereas the P-cells aged 4-5 weeks in vitro displayed a bursting activity. The qualitative morphological data evidenced abnormal swellings both on dendritic and axonal processes of the 10-11-week-old P-cells which were not present on the 4-5-week-old P-cells. The quantitative data revealed a significant decrease in the overall size of the dendritic network of the 10-11-week-old P-cells mainly due to a reduction in the total dendritic length and in the total number of dendritic segments, whereas the individual segment lengths remained almost unchanged. Dendritic spine counts showed no decrease in the dendritic density of these older P-cells. Such data suggest that the changes observed in 10-11-week-old cultured P-cells may be compared to the age-related changes occurring in vivo and that such in vitro models could be useful tools in the study of the pathology of aging. However, alternative factors other than senescence are discussed since they may account for some degenerative changes observed in the older cultured P-cells.

Animals↗
PubMed1985

Sodium butyrate-induced changes in cultured rabbit articular chondrocyte transmembrane electrical potentials. Relationship between these changes and the proliferative response.

Sodium butyrate at 5mM reversibly induced a significant increase of transmembrane potentials (Em) in normal chondrocytes (24 hours after seeding) and arrested their proliferation. This increase in Em levels, which could be temporarily abolished by Tetra-ethyl Ammonium (TEA 5mM), was related to an increase in membrane permeability to K+. This hyperpolarization was correlated with the reversible inhibition of growth in G1 induced by the sodium butyrate.

Animals↗
PubMed1985

The Purkinje cell dendritic tree: a computer-aided study of its development in the cat and in culture.

Golgi-prepared cerebella from 1, 10, 13 and 30-day-old kittens were analyzed and compared with 30-45 days in vitro (DIV) HRP-stained organotypic cultures of newborn kitten cerebella. Computer reconstructions and morphometric parameters allowed a quantitative analysis of the Purkinje cell (P-cell) dendritic trees. In intact animals the dendritic organization appeared monoplanar as early as one day after birth and biplanar in 85% of the cells at day 13; however, by day 30, 90% of the cells were monoplanar. During the first 4 postnatal weeks, the dendritic expansion was due mostly to an increase in the total number of segments and the total dendritic length, whereas the overall mean segment length remained almost unchanged. In culture, the 30-45 DIV P-cell dendritic trees always appeared reduced in size when compared to their in vivo counterparts due mostly to a reduction in the total number of segments. Nevertheless, these cells retained several primary dendritic trunks and their overall mean segment length was longer. These supposedly 'mature' cultured P-cells never reached full adult development: a discriminant analysis classified them as resembling those from intact animals of 13 days but often maintaining some properties of newborn animals. These results demonstrate that the presence of all normal inputs is required to achieve the full elaboration and the planar disposition of the P-cell dendrites.

Animals↗
PubMed1983

The dendritic trees of Purkinje cells: a computer assisted analysis of HRP labeled neurons in organotypic cultures of kitten cerebellum.

Purkinje cell dendritic arborizations were studied in HRP intracellularly stained mature neurons grown during 30-50 days in organotypic cerebellar cultures from newborn kittens. The effects of afferent fiber depletions on the final dendritic topological parameters were investigated by computer assisted methods. Three differently deafferented models lacking both parallel and climbing fibers (PF and CF), only PF or only CF were thus studied. Qualitative data were found to be common to these 3 in vitro models and similar to those following other in vivo deafferenting procedures. Quantitative data obtained from 16 measurable parameters showed that all dendritic trees were markedly reduced in size as indicated by a decrease in total dendritic length and total number of segments although in all models individual segment lengths remained largely unaltered. A discriminant analysis permitted the recognition of 3 populations (100% well classified cells) based on 3 selected variables: dendritic field area, mean path length and total number of segments (with the lowest values for the model lacking only PF). These results are shown to lend further support to the role played in the final dendritic arrangement by both intrinsic (non-discriminant) and extrinsic (discriminant) factors and more precisely by the two distinct specific afferent systems (PF and CF).

Afferent Pathways↗
PubMed1981

A simple device for making a standard inverted phase-contrast microscope movable.

A simple, useful and inexpensive device was designed and tested which allowed, under visual control at high magnification, large areas of a culture to be scanned without removing the microelectrodes inserted into remote parts of the nervous tissue. Such a device consisted of making removable, over several millimeters, an inverted phase-contrast microscope without disrupting either the preparation or the microelectrodes. This was realized by fixing the stage to a rigid metallic framework. At the same time, the optic portion of the microscope could slide smoothly on a base consisting of a pair of mirrors separated by greased, precision ball-bearings. All the focusing and scanning movements were achieved by the original rack and pinion controls of the microscope.

Animals↗
PubMed1980

[Evidence of autapses in vitro by electron microscopy combined with iontophoretic intracellular injection of peroxidase].

The existence of autapses, i. e. of synapses between a neuron and its own axon, is demonstrated in vitro by means of Horseradish peroxidase (HRP) iontophoretic filling, combined with electron-microscopic (EM) analysis. After the injection of one single Purkinje cell per explant (Kitten cerebellum), the ultrastructural observation (serial thin sections) reveals the presence of both pre- and post-synaptic labelled structures. Axodendritic autapses are thus illustrated and their significance is discussed in view of earlier morphological and electrophysiological data.

Animals↗