Morphology of vomeronasal organ cultures from fetal rat

The vomeronasal organs (VNOs) of rats were cultured from embryonic 15-day littermates on collagen-coated membrane in Dulbecco's modified Eagle's medium containing serum. The explants were observed sequentially and fixed at 4, 6, 8, 10 and 14 days in vitro (DIV). Organogenesis of VNOs and cell differentiation took place in vitro. Patterns of organogenesis of the VNO in vitro were different from those in vivo. Both sensory and supporting cells in the sensory epithelium had microvilli on their surface. Epithelial cells in aggregates of non-sensory epithelial cells had cilia and microvilli on their surface. Vomeronasal axons forming two to three large fascicles were seen originating from the VNO at 4, 6, and 8 DIV, and degenerated at 10 or 14 DIV. Glial cells (ensheathing cells) were observed in the fascicles. These morphological characteristics of VNO cells in vitro were similar to those observed in vivo.     

Ultrastructural organization of slice cultures from rat visual cortex

Summary We have been studying the fine structural organization of slice cultures prepared from the visual cortex of 6-day-old rats and cultured for 2 weeks using a roller culture technique. Neurons in culture exhibited the characteristic cytological differences between perikarya, axons and dendrites. Neuronal and glial processes formed a dense neuropil with minimal extracellular spaces, and within the neuropil there were numerous synaptic contacts. Both morphological types of cortical synapses, type I (asymmetrical) and type II (symmetrical) could be readily identified in slice cultures. The pattern of synaptic connections in culture was remarkably similar to that observed in normal cerebral cortex: asymmetrical synapses were usually found in contact with dendritic spines, less frequently with dendritic shafts, and never on perikarya, whereas symmetrical synapses were found mostly on perikarya, occasionally on dendritic shafts but never on dendritic spines. Synaptic morphology appeared mature after 2 weeksin vitro and did not show the immature features observed at the time of culture preparation. Taken together with our previous light microscopic studies, these results indicate that cortical slice cultures are organotypically organized and serve as a useful model to study mechanisms of cortical development and plasticity.     

Progesterone increases oligodendroglial cell proliferation in rat cerebellar slice cultures

We have previously demonstrated that progesterone significantly increases the rate of myelination in organotypic slice cultures of 7-day-old rat and mouse cerebellum. Here, we show that progesterone (20microM) stimulates the proliferation of oligodendrocyte precursors in cultured cerebellar slices of 7-day-old rats. The steroid increased the number of pre-oligodendrocytes (NG2(+), O4(+)) and to some extent of oligodendrocyte precursors, corresponding to an earlier developmental stage (nestin(+), PDGFalphaR(+), NG2(+), O4(-)). Progesterone stimulated the proliferation of both NG2(+) and O4(+) cells as shown by increased double-immunolabeling with the cell proliferation marker Ki67. The mitogenic effect of progesterone was inhibited by the progesterone receptor antagonist mifepristone (10microM) and could not be mimicked by its GABA-active metabolite 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone), even at the high concentration of 50microM. Results indicate that progesterone first strongly and transiently stimulates the proliferation of oligodendrocyte precursors, and that it may thereafter accelerate their maturation into myelinating oligodendrocytes. Although oligodendrocyte precursors may be a direct target for the actions of progesterone, their number may also be indirectly influenced by the effects of the steroid on neurons and microglial cells, since treatment of the cerebellar slices with progesterone enhanced staining of the neuronal cytoskeleton marker microtubule-associated protein-2 and increased the number of OX-42(+) microglia. A small percentage (about 0.1%) of the NG2(+) cells transiently became OX-42(+) in response to progesterone. These results point to novel mechanisms by which progesterone may promote myelination in the CNS, specifically by stimulating the proliferation and maturation of oligodendrocyte precursors into myelinating oligodendrocytes.     

Cellular organization and development of slice cultures from rat visual cortex

Summary Slice cultures from the visual cortex of young rats were prepared using the roller culture technique (Gähwiler 1984). After 10 days in vitro the cortical cultures flattened to 1–3 cell layers, surviving for up to 12 weeks. The cultures were organotypically organized, the typical layered structure of the cortex was preserved. The neuronal composition of slice cultures was studied using intracellular staining, Golgi impregnation and GABA immunohistochemistry. Both pyramidal cells and several types of nonpyramidal cells were identified in the slice cultures. Electrophysiological recordings showed that the electrical properties of cells in culture were similar to those measured in acute slice preparations; for some cells, however, the spontaneous activity was higher. The maintained activity was strongly increased by application of the GABA antagonist bicuculline and decreased by GABA, suggesting that GABAergic inhibition is present in these preparations. We could observe the postnatal maturation of some characteristic morphological features in culture. For example, pyramidal cells in 6 day-old rats in situ have very short basal dendrites with growth-cones, and the dendrites are free of spines. After 2–3 weeks in culture growth-cones were no longer observed. Instead, the cells had developed a large basal dendritic field and the dendrites were covered with spines. Slice cultures therefore may provide a useful tool for physiological, anatomical, pharmacological and developmental studies of cortical neurons in an organotypical environment.     

Enrichment of unipolar brush cell-like neurons in primary rat cerebellar cultures

Unipolar brush cells are a distinct class of excitatory interneurons situated in the granular layer of the cerebellar cortex, where they form giant synapses with individual mossy fiber terminals. We have previously shown that primary cerebellar cell cultures from embryonic and postnatal rodents contain neurons displaying morphological and chemical phenotypes characteristic of unipolar brush cells in situ, including intense staining with calretinin antiserum. In cultures from both embryonic and postnatal rats, the large majority of calretinin-positive neurons are unipolar brush cells, while granule cells are usually calretinin-negative. A small percentage of putative Golgi/Lugaro cells also express calretinin. We demonstrate here that the developmental stage of the source tissue, the concentration of potassium in the medium, and treatment with glutamate after differentiation have substantial effects on the density of putative unipolar brush cells in the cultures. In dissociated cultures obtained from embryos at gestation day E18 and E20 and from pups at postnatal day P0, P2, P5, P8, and P10 grown in 25 mM KCl, the percentage calretinin-positive cells progressively decreases from 24% to 0.1% of total cells. In cultures from E20 embryos grown in physiological potassium (5 mM KCl), calretinin-positive cells are enriched to approximately 60% of total cells, while the majority of calretinin-negative cells die. In embryonic cultures exposed to high concentrations of glutamate after 12 days in vitro, calretinin-positive neurons have a survival advantage over calretinin-negative cells and represent up to 83% of total cells.     

Autoradiographic localization of3H-GABA and3H-muscimol binding in rat cerebellar cultures

Summary Autoradiographic studies were conducted on the binding of³H-GABA and³H-muscimol in cultures of rat cerebellum. Binding sites for both substances were observed on many cerebellar neurones, such as Purkinje cells and interneurones, but not on glial cells. Binding of³H-GABA and³H-muscimol was inhibited by unlabelled GABA and by the GABA antagonist bicuculline.     

Freeze-fracture organization of chromatin and cytoplasm in neurons and astroglia of rat cerebellar cortex

Summary The cytology of the cell nucleus and cytoplasm of neurons and astroglia of the rat cerebellar cortex has been investigated by freeze-fracture electron microscopy. The main differential characteristics in the cytoplasm of the several cell types of the cerebellar cortex were: (1) the organization of endoplasmic reticulum elements, including special configurations of lamellar bodies and hypolemmal complexes, (2) the polarity, extension and arrangement of Golgi cisterns and associated tubulovesicular elements; (3) the connection pattern among different membrane-bounded cellular compartments; and (4) the architecture of endomembranes (i.e. presence of pits and fenestrations). In the nucleus, the main differential features were the the three-dimensional view of the nuclear envelope, the distribution of nuclear pores and the aggregation pattern of chromatin, visualized as clusters of nuclear particles in cross-fractures. The quantitative analysis of chromatin revealed four peaks of nuclear particle sizes (8, 12, 17 and 21 nm) that may correspond to variable degrees of coiling of the polynucleosomal chain in the chromatin fibre. Significant differences were observed in the proportion, numerical density and size distribution of aggregated nuclear particles in heterochromatin domains among the different cell types of the cerebellar cortex. The percentage of nuclear particles in aggregates varied from 10% in Purkinje cells to 64% in granule cells. Astrocytes and Bergmann glia showed intermediate values (about 40%). The percentage of nuclear particles in aggregates snowed a significant (P < 0.05) negative linear correlation with the nuclear volume, the number of pores per unit nuclear volume and the total number of pores per nucleus. In granule cells and astroglia, heterochromatin domains had a greater percentage of large nuclear particles (>10 nm) than did euchromatin domains, whereas in interneurons, Purkinje and Golgi cells heterochromatin and euchromatin showed a similar proportion of large particles. Nuclear particles in euchromatin exhibited a similar pattern of distribution in all cerebellar cells.     

Short-term effects of kainic acid on rat cerebellar cells in monolayer cultures

Monolayer cultures of postnatal day 5 rat cerebellar cells were grown for three weeks in Dulbecco's Modified Eagle's Medium, supplemented with either 10% horse serum or a defined hormone mixture. Sensitivity to kainic acid (10^?4 M) was monitored both morphologically and by uptake of [³H]GABA (with and without the glial uptake inhibitor β-alanine). Morphological changes could be detected after only 30 min of exposure to the kainic acid. After 4 h, granule cells appeared intact, while specific neuronal GABA uptake was about half that of untreated controls.     

Nicotinamide and 1-methylnicotinamide reduce homocysteine neurotoxicity in primary cultures of rat cerebellar granule cells

Nicotinamide is an important cofactor in many metabolic pathways and a known neuroprotective substance, while its methylated product, 1-methylnicotinamide, is a suspected neurotoxin. Homocysteine is a risk factor in Alzheimer's disease and neurodegeneration, causing inhibition of methylation processes and inducing excitotoxicity. In this study, using primary cultures of rat cerebellar granule cells and propidium iodide staining, we investigated the neurotoxicity of nicotinamide and 1-methylnicotinamide, and their neuroprotective potential in acute and sub-acute homocysteine neurotoxicity. Our results demonstrated that nicotinamide and 1-methylnicotinamide applied for 24 h to cultures at concentrations of up to 25 mM had no effect on neuronal viability. Moreover, nicotinamide at concentrations of 5-20 mM and 1-methylnicotinamide at 1-10 mM applied to cells 24 h before, and for 24 h after an acute 30 min application of 25 mM D,L homocysteine, reduced neuronal damage. 1-Methylnicotinamide at concentrations of 250 and 500 muM showed neuroprotective activity during a sub-acute 24-h exposure to 2.5 mM D,L-homocysteine, while 5 and 25 mM nicotinamide also evoked neuroprotection. These findings do not support suggestions that 1-methylnicotinamide may act as an endogenous neurotoxic agent; rather, they indicate the neuroprotective ability of nicotinamide and 1-methylnicotinamide in homocysteine neurotoxicity. The exact mechanisms of this neuroprotection are unclear and require further investigation.     

Morphological and functional organization of cortico-nuclear cerebellar projections

A neurohistological and electrophysiological study of the cortico-nuclear connections of the cerebellum confirmed the medico-lateral principle of its zonal organization. Overlapping between neighboring longitudinal zones was less marked in electophysiological experiments. Convergent and divergent relationships were found between axons of Purkinje cells to nuclear neurons both within the same zone and between different zones of the cerebellum. Cortico-subcortical influences, occasioned by the wider structural organization of the longitudinal cortico-nuclear projections, show great diversity.     

Selective induction by glucocorticoids of tyrosine hydroxylase in organ cultures of rat pheochromocytoma

Addition of physiological concentrations of glucocorticoids to organ cultures of a transplantable rat pheochromocytoma resulted in an increase in the specific activity of tyrosine hydroxylase. The increase was delayed, beginning after 12 h and reached its peak after 36 h, when the specific activity was twice that of controls. The activities of dopamine β-hydroxylase and dopa decar?ylase showed no significant changes. The tumour contains choline acetyltransferase and the specific activity of this enzyme was reduced by 25% following culture in the presence of glucocorticoids. Addition of cholinergic agonists and/or nerve growth factor to the culture medium had no effect on the specific activity of tyrosine hydroxylase. The effect of glucocorticoids on this enzyme was entirely abolished by inhibitors of ribonucleic acid or protein synthesis.These results are discussed in relation to the modulatory role of glucocorticoids in trans-synaptic and nerve growth factor-mediated enzyme induction in the peripheral sympathetic nervous system.     

A Golgi analysis of neuronal organization in the medial cerebellar nucleus of the rat.

The neurons of the medial cerebellar nucleus of the rat were studied with several variants of the Golgi technique. Using the criteria of somatic size, dendritic pattern and location, seven cell types were designated within the nucleus. Large multipolar neurons were most prominent in dorsal portions of the nucleus while small multipolar and fan-shaped neurons predominate ventrally. The rostral and caudal halves of the medial nucleus differ in their neuronal orientation. There is a caudolateral to rostromedial cellular orientation in the caudal portion of the nucleus, and a caudomedial to rostrolateral orientation in the rostro-ventral part of the medial nucleus. The large multipolar neurons of the rostro-dorsal region display no special orientation. In addition to these rostral and caudal architectural differences, there are several longitudinal zones within the medial nucleus previously reported in Nissl preparations (Beitz &; Chan-Palay, 1978). The medial and lateral elliptical zones are comprised predominantly of neurons of the nuclear boundary, fan-shaped neurons and small bipolar neurons. The round cell central zone, by contrast, is comprised primarily of small and large multipolar neurons and bouquet neurons.We conclude that the medial cerebellar nucleus consists of several distinct regions each with a characteristic arrangement of its constituent cells and that these regions correspond to those previously reported as having different physiological functions.     

Growth-stimulating action of some nitroso compounds on organ cultures of mouse and rat embryonic liver

The transplacental and direct action of nitrosomethylurea on organ cultures of the liver from 18–20-day CBA and C57BL mouse embryos and of diethylnitrosamine on liver cultures from noninbred rat embryos was studied. The nitroso compounds accelerated adaptation of the explants, increased the viability of the cultures compared with normal, and led to hyperplastic proliferation of the small basophilic cells whose survival rate under both normal and experimental conditions was higher than that of ordinary embryonic hepatocytes. The growth-stimulating effect depended on the object (species, line of animals) and the factor to which it was exposed (carcinogen, mode of administration).Department of Carcinogenic Agents, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 12, pp. 716–719, December, 1979.