Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures

Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15–16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 μM) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 ± 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 ± 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD₂ ? PGF_2α > TXB₂ ? PGE₂) and astroglial cells (PGD₂ > TXB₂ ? PGF_2α ? PGE₂ ? 6-ketoPGF_1α). Preincubation with arachidonic acid (1 μg/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4–6-fold by addition of the calcium ionophore A23187 (1 μM). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.     

Immunocytochemical localization of protein kinase C in developing brain tissue and in primary neuronal cultures

Antisera to protein kinase C (PKC) have been used to examine the presence and distribution of the enzyme in developing cerebellar cortex of postnatal rat and in cultures of rat sympathetic ganglia. In the cerebellar cortex of 2-,4-, and 6-day old rats, immunostaining was observed in areas of early-forming presynaptic terminals and growth cones. No staining was evident in the cortical proliferative zone. Beginning at 10 days postnatal, nuclear staining, not apparent at earlier stages, was prominent in Purkinje cells. In neuronal cultures of dissociated rat sympathetic ganglia, PKC was immunolocalized in cell bodies and bundles of neuronal processes. Immunoreactivity was particularly striking in growth cones of extending neurites and in axonal varicosities. These results suggest a role for PKC in neuronal growth following cell proliferation and in synaptic function. The appearance of nuclear staining in later developmental stages suggests that the enzyme may be involved in the promotion and maintenance of the differentiated state of neurons.     

Cerebellar granule cells show age-dependent migratory differences in vitro

Developmental differences between cerebellar granule cells during their migratory period were revealed using dissociated granule cell cultures isolated from 4, 7, or 10 days old (P4, P7, P10) mice. Under all culture conditions, the great majority of cultivated cell populations consisted of those granule cells that had not reach their final destination in the internal granule cell layer (IGL) by the age of isolation. In vitro morphological development and the expression of migratory markers (TAG-1, astrotactin, or EphB2) showed similar characteristics between the cultures. The migration of 1008 granule cells isolated from P4, P7, and P10 cerebella and cultivated under identical conditions were analyzed using statistical methods. In vitro time-lapse videomicroscopy revealed that P4 cells possessed the fastest migratory speed while P10 granule cells retained their migratory activity for the longest time in culture. Cultures obtained from younger postnatal ages showed more random migratory trajectories than P10 cultures. Our observations indicate that despite similar morphological and molecular properties, migratory differences exist in granule cell cultures isolated from different postnatal ages. Therefore, the age of investigation can substantially influence experimental results on the regulation of cell migration.     

Cerebellar granule cells show age‐dependent migratory differences in vitro

Developmental differences between cerebellar granule cells during their migratory period were revealed using dissociated granule cell cultures isolated from 4, 7, or 10 days old (P4, P7, P10) mice. Under all culture conditions, the great majority of cultivated cell populations consisted of those granule cells that had not reach their final destination in the internal granule cell layer (IGL) by the age of isolation. In vitro morphological development and the expression of migratory markers (TAG‐1, astrotactin, or EphB2) showed similar characteristics between the cultures. The migration of 1008 granule cells isolated from P4, P7, and P10 cerebella and cultivated under identical conditions were analyzed using statistical methods. In vitro time‐lapse videomicroscopy revealed that P4 cells possessed the fastest migratory speed while P10 granule cells retained their migratory activity for the longest time in culture. Cultures obtained from younger postnatal ages showed more random migratory trajectories than P10 cultures. Our observations indicate that despite similar morphological and molecular properties, migratory differences exist in granule cell cultures isolated from different postnatal ages. Therefore, the age of investigation can substantially influence experimental results on the regulation of cell migration. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Vitamin E affects cell death in adult rat dentate gyrus

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.     

Vitamin E regulates acetylcholine receptor function of molluscan neurons.

1. Intracellular recordings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia. 2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recording and voltage-clamp techniques. 3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10(-6) to 10(-4) M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10(-5) to 5.10(-5) M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current. 4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective. 5. The calmodulin inhibitor, chloropromazine (6.10(-5) M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E. 6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found. 7. Application of 10(-4) to 10(-6) M vitamin E (DL-alpha-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon. 8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.     

Development of monoamine oxidase activity and monoamine effects on glutamate release in cerebellar neurons and astrocytes

Activities of monoamine oxidase (MAO) A and B were measured during the first month of postnatal development in mouse cerebellum and in primary cultures of either cerebellar granule cells or cerebellar astrocytes, derived from 7-day-old cerebella. In addition, effects of the two monoamines, serotonin (a MAO A substrate) and phenylethylamine (a MAO B substrate) on the release of glutamate under resting conditions and in a transmitter related fashion (i.e., potassium-induced, calcium-dependent glutamate release) were studied during the same period. Both MAO A and MAO B activities increased during in vivo development (beginning around postnatal day 14) and in cultured astrocytes (during a comparable time period and to a similar extent), but remained constant at a low level in granule cells. In 4-day-old cerebellar granule cell cultures there was no potassium-induced glutamate release but serotonin as well as phenylethylamine reduced the release in both the presence and absence of excess potassium. In 8- and 12-day-old granule cell cultures and in 8- and 18-day old astrocyte cultures there was a pronounced glutamate release during superfusion with 50 mM K⁺. In both neurons and astrocytes this response was inhibited by 1 nM of either serotonin or phenylethylamine. In the astrocytes the inhibition was followed by an increased release of glutamate in both the presence and absence of the high potassium concentration, whereas the 8-day-old neurons showed only a slight increase in glutamate release after the with-drawal of the monoamine and only in the absence of excess potassium. The response was almost identical in 8-and 18-day-old astrocytes in spite of the marked difference in MAO activities.Special issue dedicated to Dr. Paola S. Timiras.     

An early increase in the disialoganglioside GD3 contributes to the development of neuronal apoptosis in culture

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.     

Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures [corrected

Boar sperm from the proximal caput epididymis were co-incubated with 1, 4, 7, 10 and 14-day old caput, corpus and cauda epididymal cultures for 24, 48 and 72 h. Boar kidney epithelial cells (LLC-PK1) and ECM alone were used as negative controls. Sperm motility, morphology and membrane integrity were studied to evaluate boar sperm maturation in vitro. Our results showed that epithelial cell monolayers (10, 14-day old) create a suitable microenvironment for the survival of proximal caput sperm and the maintenance of sperm motility over a 72 h period. Moreover, corpus epididymal tubule fragments in culture (1, 4-day old) are capable of promoting the migration of the cytoplasmic droplet along the sperm tail after 24h of co-incubation.     

Protective effect of vitamin E on reduction in colony formation of cultured human cells by bisulfite

Bisulfite had a severe cytotoxic effect on HeLa S-3 cells at a concentration of 10⁻⁴ M, causing complete inhibition of colony formation. A concentration of 10⁻M bisulfite had a moderate cytotoxic effect on the cells, reducing their colony-forming activity to one-third of that of untreated control cultures. Addition of vitamin E at a concentration of 10⁻⁷ M restored the colony-forming activity of 10⁻⁵ M bisulfite-treated cells to 71% of that of control cultures. Addition of 10⁻⁶ M vitamin E resulted in almost complete recovery of the colony-forming activity of bisulfite-treated cells to that of the control cultures. The counteraction of vitamin E to the cytotoxic effect of bisulfite on human cells was discussed.     

Co-cultivation of astroglial enriched cultures from striatum and neuronal containing cultures from substantia nigra

A co-cultivation system was developed with neuron-containing (neuron-specific enolase (NSE) positive) primary cultures from the substantia nigra of 15 to 17-day old embryonic rats which were grown 1 mm apart from astroglial-enriched (glial fibrillary acidic protein (GFAp) positive) primary cultures from the striatum of neonatal rats. The astroglial cells went through a morphological differentiation with extension of processes after co-cultivation with the immunohistochemically-identified neuronal cells. The astroglial-enriched striatum cultures showed a higher active uptake of 3H-L-glutamate after co-cultivation for one week, compared to control cultures from striatum. Vmax (nmol X mg protein-1 X min-1 X was 58.4 +/- 8.3 after co-cultivation and 37.2 +/- 6.3 for control cultures. The glutamine synthetase (GS) activity was slightly increased after co-cultivation. The validity and specificity of the results were ensured. The data suggest that astroglial cells in a primary culture are influenced by co-cultivation with fetal neuron containing cultures resulting in morphological differentiation, and increases in 3H-L-glutamate uptake and GS activity.     

Effect of culture conditions on monoclonal antibody production from genetically modified tobacco suspension cultures

Oxygen supply and inoculum age were found to affect the production of the heavy chain monoclonal antibody (HC MAb) from genetically modified tobacco suspension cultures. The increase of oxygen supply increased both cell growth and HC MAb production. Furthermore, the increased aeration and mixing improved the production of HC MAb based on the unit amount of cells or total soluble proteins. This indicated that the increased aeration improved the production and secretion of HC MAb more than other cell components. HC MAb production and cell growth also improved when batch cultures were inoculated with actively dividing cells (5-day old) rather than the fullygrown cells (7- or 10-day old cells) that are commonly used for subcultures. The addition of glutamine to the medium also improved cell growth and HC MAb production.     

Molecular Forms of Acetylcholinesterase in Pineal Cell and Sympathetic Neuronal Cultures

Abstract The activities of the various molecular forms of acetylcholinesterase (AChE) were measured in monolayer cultures of neonatal rat pineal cells grown alone and in co-culture with sympathetic neurons. AChE forms characterized by sedimentation coefficients of 4S, 6.5S, and 10S were found in the neuronal and pineal cultures, as well as in the co-cultures. The 16S AChE form was found only in the neuronal cultures. Total AChE activity increased with culture age in the co-cultures, but it decreased in pineal cells cultured alone. The low level of activity present in the neuronal cultures did not change markedly over the 27-day culture period. These results, which show bidirectional neuron-pineal cell effects, suggest that AChE molecular forms may be important markers to study the mechanisms underlying neuron-target cell interaction in the developing sympathetic nervous system.     

The effect of donor age on the sensitivity of osteoblasts to the proliferative effects of TGF(beta) and 1,25(OH(2)) vitamin D(3)

The loss of osteoblast function in aging bone is one of the major causes of osteopenia, or loss of bone mass. In this study, this loss of function was investigated by examining the proliferative response of rat long bone periosteal osteoblasts to TGF(beta1) and 1,25-dihydroxy vitamin D(3) (1,25-D(3)) as a function of donor age. Using a DNA binding fluorescent dye, DNA levels were measured in osteoblast cultures derived from either young adult (3-4 months) or old (14-15 months) rats following treatment with two concentrations (10(-9) M or 10(-12) M) of either 1,25-D(3) or TGF(beta1) or with vehicle. Cells from young rat bone, when treated with 1, 25-D(3), showed a dose-dependent increase in proliferation when treated with the higher dose and a decrease in proliferation when treated with the lower dose. Osteoblasts isolated from old rats did not respond to 1, 25-D(3) treatment. A similar pattern of response to TGF(beta1) was found. When treated with 10(-9) M TGF(beta1), the rate of proliferation increased for young rat osteoblasts, but the old rat derived cells were unresponsive. The 10(-12) M dose of TGF(beta1) was ineffective for both young and old cells. This study has shown that osteoblasts derived from old donors are impaired in their ability to respond to vitamin D and TGF(beta), two of the major controlling factors of skeletal development and maintenance.     

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.     

Differential response among cells in the chick embryo myogenic lineage to photosensitization by Merocyanine 540

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.