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Biomedical subjects

H J Romijn

Publications and source records attributed to H J Romijn.

At least 37 records · Page 2Linked to original sources
PubMed1989

Preferential loss of GABAergic neurons in hypoxia-exposed neocortex slab cultures is attenuated by the NMDA receptor blocker D-2-amino-7-phosphonoheptanoate.

The preferential loss of GABAergic neurons in neocortex slab cultures after exposure to hypoxia was reinvestigated and confirmed using an improved morphometric analysis based on stereological principles. This preferential cell loss could be attenuated by treating the hypoxic slab cultures with the N-methyl-D-aspartate (NMDA) receptor blocker D-2-amino-7-phosphonoheptanoate (D-APH).

2-Amino-5-phosphonovalerate↗
PubMed1989

Unlike hypoxia, hypoglycemia does not preferentially destroy GABAergic neurons in developing rat neocortex explants in culture.

We tested whether hypoglycemia, like hypoxia, would preferentially destroy GABAergic nerve cells in the neocortex. To this end, rat neocortex explants dissected from 6-day-old rat pups and cultured up to a developmental stage approximately comparable to that of the newborn human neocortex, were exposed to hypoglycemia for different periods. Quantitative light microscopic and immunocytochemical evaluation of the cultures demonstrated that hypoglycemia does not preferentially destroy GABAergic but rather non-GABAergic neurons, a finding quite opposite to what was found after hypoxia. Recent biochemical data from other laboratories which seem to support this difference in neuronal vulnerability are discussed. It is concluded that perinatal hypoglycemia may not form such a serious threat with respect to the genesis of epilepsy as does hypoxia.

Animals↗
PubMed1988

Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture.

The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a developmental stage approximately comparable to that of the newborn human neocortex were exposed to hypoxia for different periods. Quantitative light microscopic and immunocytochemical evaluation of the cultures demonstrated that GABAergic neurons were indeed the first neurons to die during hypoxia.

Animals↗
PubMed1988

A procedure for culturing rat neocortex explants in a serum-free nutrient medium.

A procedure is described for long-term culturing of rat neocortex explants in a serum-free growth medium. Slices spanning the entire cortical depth from pial to ventricular side are prepared from 6-day-old rat pups. After preincubation in Hanks' balanced salt solution with extra glucose, the explants are placed on polyamide gauze carriers in plastic culture dishes containing serum-free medium. The dishes are continuously rocked during the culture period. After 3 weeks in vitro the explants consist of a three-dimensional network of neural tissue with a mean thickness above the gauze of ca. 100 micron which corresponds with about 8 cell layers. Central necrosis is either fully absent (in one-third of the explants) or restricted to a minimal strip or patch located close to the gauze. From pial to ventricular side, 5 layers can be distinguished which, with respect to cell size and cell density, reflect a histiotypic architecture. The dense neuropil shows abundant axo-dendritic synapses (both on shafts and spines), myelinated fibers, and spontaneous bioelectric activity.

Animals↗
PubMed1988

Development and advantages of serum-free, chemically defined nutrient media for culturing of nerve tissue.

This paper describes in a historical perspective the development of serum-free nutrient media suitable for long-term culturing of nerve tissue. Several disadvantages of the use of serum are discussed, coupled with an acknowledgement that it is not always advisable to replace a routinely used serum-supplemented medium by a chemically defined medium with the expectation of immediate success. Therefore a strategy is given on how to develop a chemically defined medium that is thoroughly tuned to the specific needs of the cell type to be cultured. It is argued that such a medium has several substantial advantages over the use of serum.

Animals↗
PubMed1988

Cytoarchitecture in cultured rat neocortex explants.

Neocortex explants obtained from 6-day-old rat pups and cultured in a serum-free medium from 5 hr to 13 days in vitro (DIV) show preservation of cytoarchitectural characteristics. Major changes in the size of the explants and their layers occur during the first 2 DIV. A radial arrangement of neurons within layer 2-3-4, which becomes apparent between 2 and 10 DIV, suggests an advance in maturation in culture. In contrast to the situation in vivo, a distinct layer 4 cannot be consistently identified. During the first 2 DIV, a transposition of cells into the pial direction can be seen. Individual degenerated cells are spotted especially in layer 5. The presence of these cells amidst healthy neurons suggests that their death has a functional cause. Neurons at the ventricular border of layer 6 become relatively large in comparison with other neurons. Within the cultured tissue there is a marked increase in GFA reactivity compared to the situation in vivo. The described results clearly indicate that in these cultured explants, both similarities and differences are of interest for studies on the formation of neuronal circuitry within the cerebral cortex.

Animals↗
PubMed1987

Tetrodotoxin enhances initial neurite outgrowth from fetal rat cerebral cortex cells in vitro.

The effect of two different concentrations of the sodium channel blocker tetrodotoxin on neurite outgrowth from fetal rat cerebral cortex neurons was studied in vitro. A concentration of 10(-6) M tetrodotoxin enhanced after two days in vitro: the percentage of isolated cells and reaggregates which formed neurites, the number of neurites per cell soma, and neurite elongation and subsequent branching. The effects observed after treatment with 10(-7) M tetrodotoxin were on the whole intermediate between control and 10(-6) M tetrodotoxin.

Animals↗
PubMed1987

Postembedding immunocytochemical GABA labeling in rat neocortex cultures: applicability in quantitative studies.

The value of gamma-aminobutyric acid (GABA) immunogold labeling in the quantification of nerve endings was studied in dissociated rat neocortex cultures. Adjacent sections were processed in 3 series according to the same staining protocol. Of all nerve ending profiles examined, 20% appeared to be GABA-positive, while 7% could not be classified. In cultures pretreated with gabaculine, GABA-positive endings were labeled more heavily. Cell bodies could always be discriminated as GABA-positive or GABA-negative. Of all neuronal cell profiles, 14% appeared to be GABA-positive. These neurons could also be identified by light microscopy using the peroxidase-anti-peroxidase method.

Animals↗
PubMed1987

Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin.

Our aim was to determine if chronic blockade of GABAergic inhibitory synaptic activity, monitored electrophysiologically at the neuronal level, would affect synapse formation and ultrastructure in dissociated fetal rat cerebral cortex cultures. This was achieved by adding picrotoxin to the serum-free growth medium in a dose that induced continuous epileptiform discharges throughout the culture period. Light and electron microscopic analysis suggested an accelerated synaptic network formation in the experimental cultures during the first 2 weeks in vitro. The elimination of excess synapses (mainly on spines), which normally takes place during the fourth week in vitro, occurred 1 week earlier in the presence of picrotoxin. Finally, the experimental cultures showed smaller spine synapses throughout the entire culture period. Because these effects were opposite those induced by chronic tetrodotoxin-blockade of spontaneous bioelectric activity in a previous study, the underlying causal factor could be the respective intensification and suppression of neuronal activity in the two experiments. An appropriate balance between excitatory and inhibitory synaptic drive seems therefore to be important for normal maturation of neocortical circuitry.

Action Potentials↗
PubMed1987

Indications for a critical period for synapse elimination in developing rat cerebral cortex cultures.

It was observed in an earlier study that chronic tetrodotoxin (TTX) blockade of spontaneous bioelectric activity (SBA) in rat cerebral cortex cultures prevented the large-scale elimination of synapses which normally occurs during the fourth week in vitro. This prompted us to study whether the persisting high synapse density during long-term TTX-treatment would still return to the "normal' low level after restoration of SBA. Therefore, cultures grown in TTX-supplemented medium for 5 weeks were switched to control medium for an additional week prior to fixation. Electron microscopic analysis showed that the numerical synapse density remained at a high level, thus suggesting the presence of a critical period whereafter bioelectrically controlled elimination of redundant connections no longer occurs. In contrast, the mean size of synaptic structures depended only on the functional state of the tissue at the moment of fixation, being larger in TTX-silenced cultures than in bioelectrically active ones regardless of treatment during the first 5 weeks in vitro.

Animals↗
PubMed1985

An improved EPTA staining method for synapses in rat cerebral cortex cultures.

Ethanolic phosphotungstic acid (EPTA) staining has proved to be a useful tool in electron microscopic studies on synapse formation and maturation in the developing brain. However, the available staining procedures did not give satisfactory contrast between synaptic structures and background in reaggregated cerebral cortex tissue grown in vitro. Such contrast is a prerequisite for reliable quantitative studies. We therefore varied several steps of the staining procedure and have succeeded in obtaining a modification which gives the required contrast. A short rinsing time after EPTA staining turned out especially to be very crucial.

Animals↗
PubMed1985

Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin.

Reaggregated occipital cortex cells of 19-day-old fetal rats were grown in a serum-free, chemically defined medium, and chronically exposed to impulse-blocking levels of tetrodotoxin (TTX) in order to study the role of bioelectric activity in synaptogenesis. As judged by phase-contrast microscopy, no differences were noticed in the development of neuronal networks in the TTX-treated vs control cultures. In addition, when TTX was withdrawn from experimental cultures at any stage of development, bioelectric activity qualitatively comparable to that of the control cultures appeared within 1 min. However, quantitative stereological EM analysis revealed a significant retardation in synapse formation and ultrastructural maturation of synaptic junctions during the first 3 weeks. Around 23 days in vitro, the central zone of the reaggregates in control cultures started to degenerate, but not earlier then day 27 in TTX-treated cultures. During this time, the control, but not the experimental cultures showed (in intact tissue regions mainly situated at the outside of the aggregates) a large and selective loss of spine synapses. It is concluded that functional blockade not only retards the early growth and maturation of synaptic networks but also prevents the later occurring selective loss of spine synapses.

Animals↗
PubMed1984

Towards an improved serum-free, chemically defined medium for long-term culturing of cerebral cortex tissue.

The present study describes a series of experiments which have led to a substantially improved serum-free, chemically defined medium (CDM) for long-term culturing of reaggregated fetal rat cerebral cortex tissue. A reduction of the original medium concentrations of the hormones insuline, T3 and corticosterone, on the one hand, and an enrichment of the medium with the vitamins A, C and E, the unsaturated fatty acids linoleic and linolenic acid, and biotin, L-carnitine, D(+)-galactose, glutathione (reduced) and ethanolamine, on the other hand, formed the most important chemical adjustments of the medium. With the aid of this CDM (encoded R12), the light- and electron microscopic architecture of the tissue could be kept in a good condition (superior to that seen earlier in serum-supplemented medium) up to 23 days in vitro. From that time on, the neuronal network lying between the reaggregates degenerated for the largest part, while a portion of the large neurons (probably pyramidal cells) plus some of the neuronal network within the reaggregates degenerated too. This degeneration process continued during the following weeks, but the reaggregates nevertheless retained most of their mass, so that both small and large neuronal cell bodies (visible in transparent regions at the edge of the reaggregates) remained in good condition up to at least 103 DIV. Stout, thick nerve bundles interconnecting the reaggregates, also survived up to this point. Electron microscopic evaluation of such 'aged' reaggregates revealed degenerating as well as healthy regions. The latter had indeed retained healthy-looking pyramidal and non-pyramidal neurons, embedded within a dense neuropil which was often traversed by myelinated axons. The numerical synapse density in such selected, healthy tissue regions reached its maximum during the sixth week in vitro, followed by a rapid decrease and a stabilization at about half the peak values. The present culture system has opened the possibility for performing controlled quantitative studies on the relationship between structure and function of cerebral cortex tissues during development and aging, on its dependence on nutrients, hormones and drugs, and on special factors synthesized by the tissue and released into the nutrient medium.

Animals↗
PubMed1984

Culture of rat cerebral oligodendrocytes in a serum-free, chemically defined medium.

Oligodendrocytes were isolated from the cerebra of young rats (5-10 days old) by trypsinization of the tissue followed by cell separation on Percoll gradients. The isolation was carried out in physiological, isotonic media. The cell yield was 2-4 X 10(6) cells per brain; the plating efficiency was greater than or equal to 70%. Isolated cells were seeded on poly-L-lysine-coated culture dishes and maintained in a serum-free, chemically defined medium for at least 30 days. After 10 days in culture 67 +/- 10% of the surviving cells were oligodendrocytes, as judged by immunocytochemical and morphological criteria, whereas most of the other cells reacted positively with antiserum against glial fibrillary acidic protein. The expression of typical oligodendrocyte markers (2':3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebrosides and myelin basic protein) was greatly enhanced under these serum-free conditions as compared with cultures in serum-containing medium. The antigenic markers (galactocerebrosides, myelin basic protein) were absent in the freshly isolated cells but could be detected after 3 days in culture by immunocytochemistry. The activity of 2':3'-cyclic-nucleotide 3'-phosphodiesterase increased from 75 nmol min-1 mg-1 protein on day 4 to 400 nmol min-1 mg-1 protein on day 14 in culture.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1981

Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures.

Co-cultures of mouse spinal cord with dorsal root ganglion (DRG) cultures were grown either in horse serum (HS)-supplemented medium or in a serum-free, chemically defined medium (CDM). The cytoarchitecture of cord--DRG explants was fully retained in CDM, with little or no distortion due to flattening of the explant, as is invariably observed in HS-supplemented cultures. Functional properties such as bioelectric activity and DRG--spinal cord interconnectivity were well sustained in CDM.

Animals↗
PubMed1981

A quantitative electron microscopic study on synapse formation in dissociated fetal rat cerebral cortex in vitro.

Occipital cortex of 19-day-old fetal rats was dissociated and cultured in vitro for 2-5 weeks in horse serum supplemented Eagle's MEM. The outgrowing neurons rapidly formed a dense network which started to degenerate after 3 weeks in vitro. By means of electron microscopy the numerical development of 6 different categories of synapses was followed during the time in culture. This approach revealed a sigmoid growth curve emerging over the first 3 weeks for the category of axo-dendritic synapses (which comprised the bulk of all synapses counted). Thereafter a decline in number set in. Chronic exposure of these dissociated cerebral cortex cultures to 50 microgram/ml xylocaine (a concentration adequate to block all measurable bioelectrical activity) did not prevent the formation of functional synaptic networks having normal synaptic ultrastructure. These results are in agreement with previous studies on fetal cerebral explants in culture. However, in some groups of our cultures, xylocaine led to a retardation in neurite outgrowth and in numerical synapse formation. Since these xylocaine effects were dose-related at a concentration double that required to silence the cultures, the growth retardation was probably not due to selective suppression of bioelectric activity, but rather to some general cytotoxic effect of the drug. In one of the xylocaine-treated groups (50 microgram/ml) which showed an exceptionally rapid neuronal outgrowth by the use of horse serum obtained from a different source than in the other groups, no deficit was noted in the number of synapses formed. Extracellular recording in this latter drug-treated group of cultures during the third week revealed (after return to control medium) spontaneous isolated action potentials, burst patterns and slow waves which were indistinguishable from the bioelectric activity seen in the control cultures. It is concluded that bioelectric activity in dissociated cortex cultures is not a prerequisite for the formation of apparently normal, functional synaptic networks.

Animals↗
PubMed1981

Nerve outgrowth, synaptogenesis and bioelectric activity in fetal rat cerebral cortex tissue cultured in serum-free, chemically defined medium.

Dissociated and non-dissociated cerebral cortex of fetal rat was successfully cultured in a serum-free, chemically defined medium for at least 18 days without any preincubation in serum-supplemented medium. Neurite outgrowth, synapse formation and spontaneous bioelectric activity, in its qualitative and quantitative aspects, were essentially the same as was observed in medium containing 20% heat-inactivated horse serum.

Animals↗