PubMed Health⌕ Search

Biomedical subjects

A Vernadakis

Publications and source records attributed to A Vernadakis.

At least 19 recordsLinked to original sources
PubMed1992

Acetyl-L-carnitine has a neuromodulatory influence on neuronal phenotypes during early embryogenesis in the chick embryo.

Studies from this laboratory and others have demonstrated that neuroblasts in early embryogenesis exhibit a high degree of plasticity with respect to neurotransmitter phenotype. The critical period within which these neuroblasts are sensitive to the effects of endogenous neurotrophins has been defined as 1-3 days of development in the chick embryo. In this study, we examined the influence of acetyl-L-carnitine (ALCAR) administered in ovo during embryonic days 1-3 (E1-E3) and sacrificed at embryonic day 8 (E8) on cholinergic and GABAergic neuronal phenotypes using as neuronal markers the activities of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), respectively. Phenotypic expression was assessed in 3 distinct anatomical regions of the embryonic brain: cerebral hemispheres (CH), optic lobes (OL), and diencephalon-midbrain-brainstem (DMBS). A single administration of ALCAR at embryonic day 1 resulted in a dose-dependent increase in ChAT activity and decrease in GAD activity in CH. ChAT activity was again increased and GAD activity decreased in CH from embryos that were administered ALCAR (100 micrograms/50 microliters/day) daily from embryonic day E1 to E3. No change was observed in either ChAT or GAD activity in OL in response to ALCAR administration during the critical period (E1-E3) at doses ranging from 10 to 500 micrograms/day. In the DMBS, the activity of ChAT exhibited a marked increase at lower doses (10 micrograms) followed by a marked decrease at higher doses (500 micrograms) of ALCAR. The decrease in ChAT activity in DMBS was again observed at an ALCAR dose of 100 micrograms/day when administered from E1 to E3.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcarnitine↗
PubMed1992

Ethanol increases cholinergic and decreases GABAergic neuronal expression in cultures derived from 8-day-old chick embryo cerebral hemispheres: interaction of ethanol and growth factors.

We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth both in ovo and in vitro. Moreover, we have found that growth factors significantly attenuate the in ovo neurotoxicity produced by ethanol. In this study, we further examined the direct effects of ethanol exposure on neuron-enriched cultures derived from 8-day-old chick embryo cerebral hemispheres consisting primarily of differentiated neurons. In addition, we examined the interaction of ethanol and nerve growth factor (NGF) or epidermal growth factor (EGF) when the growth factors were given concomitantly with ethanol. Choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) were used as markers for cholinergic and GABAergic neuronal phenotypic expression, respectively. We found that ethanol alone enhanced ChAT and reduced GAD activities in a dose-dependent manner. NGF and EGF given alone enhanced the expression of both neuronal phenotypes. When NGF was given concomitantly with ethanol at C4-8 the decline in GAD produced by ethanol was reversed. The effects of concomitant administration of ethanol and growth factors on ChAT activity revealed that ethanol interfered with the increases produced by the growth factors and especially with NGF when given alone. We conclude from these findings that ethanol may interfere with neuronal phenotypic expression by altering neuronal responsiveness to neurotrophic signals important for neuronal differentiation.

Animals↗
PubMed1992

Comparative biochemical, morphological, and immunocytochemical studies between C-6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres.

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.

Aging↗
PubMed1992

Ethanol administration during early embryogenesis affects neuronal phenotypes at a time when neuroblasts are pluripotential.

Our previous studies have reported that ethanol administration during a critical period of development profoundly affects the expression of neuronal phenotypes in whole brains of chick embryos. The present study examines a) the long-lasting effects of early ethanol treatment on neurotransmitter phenotypic expression and b) its differential effects on anatomically discrete regions of the developing chick CNS. Ethanol (10 mg/50 microliters) was administered to embryos via the air sac from E1 to E3. Embryos were sacrificed on days 4, 8, 10, or 15 of embryonic development (E4, E8, E10, E15) and assayed in specific regions of the CNS for glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT) as markers for GABAergic or cholinergic neurons, respectively. The magnitude of the developmental profile for ChAT was highest in spinal cord (SC), with similar profiles observed for cerebral hemispheres (CH) and optic lobes (OL). In contrast, the developmental profile for GAD was highest in OL and lowest in SC. Thus, neuronal phenotypes inhabit specific CNS areas from early primordial stages of development. Furthermore, cholinergic neuronal populations in discrete CNS areas reached mature levels by E10, whereas GABAergic populations continued to increase throughout the experimental period. We suggest that GABAergic precursor neuroblasts may differentiate at a later embryonic age and that specific regional factors may play a role in neuronal distribution and the rate of maturation. As reported previously, primordial CNS areas exposed to ethanol (E1-E3) exhibited a differential sensitivity. Cholinergic neuronal expression in CH remained retarded throughout the experimental period examined, whereas the early decline observed at E4 in SC cholinergic expression was reversed by E15.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1992

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age.

We have shown that in embryos treated with ethanol in ovo during days 1-3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.

Animals↗
PubMed1992

Ayurvedic (science of life) agents induce differentiation in murine neuroblastoma cells in culture.

Many Indian Ayurvedic (science of life) agents have been introduced into the U.S.A. as food supplements. Two of them, Maharishi Amrit Kalash-Ambrosia (MAK-A) and Maharishi Amrit Kalash-Nectar (MAK-N) are under investigation. This study shows that an ethanol extract of MAK-A induced morphological (neurite formation) and biochemical (increase of activity of tyrosine hydroxylase by about 15-fold) differentiation in murine neuroblastoma (NBP2) cells in culture, whereas an aqueous extract of MAK-A increased only the activity of tyrosine hydroxylase but to a much lesser extent. The treatment time of 3 days was needed for the expression of maximum differentiation. Ethanol extracts of MAK-A and aqueous extracts of MAK-A increased the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) by about 4-fold in 3 days but they did not do so in 15 min. Ethanol extracts of MAK-A also induced neurite formation in neuroblastoma cells grown in serum free medium but the concentration requirement was about a fifth of that needed in serum. The treatment time of 24 hr was sufficient to induce optimal differentiation in neuroblastoma cells grown in serum free medium. The differentiating agents in ethanol-MAK-A were resistant to heat and light and could not be removed by treatment with activated charcoal. Neither ethanol-MAK-N nor aqueous-MAK-N induced differentiation in neuroblastoma cells, suggesting that the differentiating agents were present only in MAK-A.

Adenylyl Cyclases↗
PubMed1992

Differences in neuronal and glial cell phenotypic expression in neuron-glia cocultures: influence of glia-conditioned media and living glial cell substrata.

Neuron-glia cocultures were prepared using, as a source for glial cells, either C6 glia (2B clone) of early (2B23) or late (2B111) passages or advanced passages of glial cells derived from primary cultures prepared from aged mouse cerebral hemispheres (MACH). Six-day-old chick embryo cerebral hemispheres (E6CH) were the source of neuron-enriched cultures. Glutamine synthetase (GS) activity was used as a marker for astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was used as a marker for oligodendrocytes. GS activity was markedly enhanced in cocultures of E6CH neurons and 2B23 glioblastic cells, whereas GS activity was reduced in cocultures of E6CH neurons and 2B111 astrocytic glia. In contrast, CNP activity was enhanced in cocultures of C6 glial cells with E6CH neurons. Glial cells from aged mouse brain did not respond to coculturing with E6CH neurons. It appears from these findings that neuronal input enhances the differentiation of glioblastic cells to either astrocytic or oligodendrocytic expression, whereas it decreases the activity of committed astrocytes. In contrast, glial cells from aged mouse brain do not respond to neuronal input. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, was enhanced only when E6CH cultures were grown in conditioned medium (CM) from 2B23 glioblastic cells. In contrast, ChAT activity was markedly diminished when E6CH neurons were cocultured with MACH glial cells but not when grown in CM from MACH glial cells. Thus, humoral factors from immature glial cells appear to enhance cholinergic neuronal phenotypic expression whereas cell-cell membrane contacts with aged glial cells diminish cholinergic phenotypic expression. The findings present supportive evidence that neuron-glia interrelationships are age dependent.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1992

Neuron-enriched cultures derived from spinal cord of 10-day-old chick embryos: influence of neuropeptides on neuronal survival, proliferation and cholinergic expression.

The developmental regulation of cell proliferation, survival and cholinergic expression by growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) was investigated in neuron-enriched cultures derived from 10-day-old embryonic chick spinal cord. In this study, 3H-thymidine in corporation into DNA was assessed, using two different applications, in order to determine both cellular proliferation and survival. The rate of neuroblast proliferation in both control and neuropeptide-treated cultures increased or remained the same up to day 6. However, in neuropeptide-treated cultures the magnitude of cell proliferation remained at levels higher than those observed in controls through day 6 and was most significant in SRIF-treated cultures at C4. In all groups, proliferation markedly declined by day 8. Survival of neuronal cells labelled at C4 remained high up to day 12 in all three groups, then drastically declined by day 17. Neuronal survival in the neuropeptide-treated cultures was also higher than in controls. Cholinergic expression, as assessed by activity of choline acetyltransferase (ChAT), responded differentially to neuropeptide treatment. Cultures treated with GHRH (100 nM) exhibited a long term significant enhancement in ChAT activity throughout the culture period, whereas those treated with SRIF (50 nM) expressed a transient decline in ChAT activity. Videometric analysis showed that both neuropeptides enhanced neuronal aggregation, neuritic arborization and neuritic length. These findings lead us to suggest that GHRH and SRIF may provide neurotrophic signals important not only for neuronal proliferation and survival but also for cholinergic neuronal expression. Furthermore, we propose that GHRH possesses specific cholinotrophic properties, whereas SRIF may act as a general neurotrophic factor.

Animals↗
PubMed1991

Growth hormone-releasing hormone and somatostatin influence neuronal expression in developing chick brain. III. GABAergic neurons.

We have shown that the endogenous neuropeptides, growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) influence expression of both cholinergic and catecholaminergic neuronal phenotypes in developing chick brain as assessed by the activities of choline acetyltransferase and tyrosine hydroxylase, respectively (Dev. Brain Res., 49 (1989) 275-280; Brain Research, 512 (1990) 297-303). In this study we examined the effects of GHRH and SRIF on GABAergic neuronal expression in ovo using activity of glutamate decarboxylase (GAD) as a neuronal marker. Chick embryos were administered GHRH or SRIF in ovo via the air sac on embryonic days 1, 3, 5 and 7, sacrificed at day 8 and the activity of GAD assayed in whole brain homogenates. GAD activity was significantly reduced in peptide-treated embryos as compared to controls. Similar results were obtained when GHRH was administered in a single dose at days 1 or 3 or when SRIF was administered in a single dose at day 3; GAD activity was significantly reduced as compared with control embryos. In contrast, embryos treated with either GHRH or SRIF on day 5 of development showed no difference in GAD activity as compared to controls. These data support our previous findings that endogenous neuropeptides such as GHRH and SRIF possess important properties with respect to neuronal phenotypic expression. They further define the critical period of sensitivity to these neuropeptides as 1-3 days of embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Muscle-derived factors reverse the cholinotoxic effects of ethanol during early neuroembryogenesis in the chick embryo.

The interaction between muscle-derived factors and ethanol on cholinergic neuronal expression was studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase (ChAT) as cholinergic neuronal marker. Ethanol (10 mg/50 microliters) and limb muscle extract (130 micrograms protein/50 microliters) (LME) were administered in ovo either alone or concomitantly at embryonic days 1-3 (E1-E3); or ethanol was given E1-E3 and followed by LME at E4-E7. All groups were sacrificed at embryonic day 8 (E8) and ChAT activity was assayed in homogenates of whole brain and of spinal cord. As previously reported, ethanol at E1-3 produced a 30% decrease in brain ChAT activity and 35% in spinal cord. Concomitant administration of ethanol and LME at embryonic days E1-E3 eliminated the decrease in choline acetyltransferase activity produced by ethanol in the brain, but not in the spinal cord. On the other hand, administration of LME at embryonic days E4-E7 to embryos pretreated with ethanol at embryonic days E1-E3, raised ChAT activity to control level in the spinal cord, but only partially restored ChAT activity in the brain. In view of the alleged neurotrophic effects of muscle-derived factors on neuronal survival and neuronal growth, we interpret these findings to suggest that LME in addition to its ability to decrease natural neuronal death, may prevent death resulting from neurotoxicity.

Animals↗
PubMed1991

Effects of early in ovo administration of ethanol on expression of the GABAergic neuronal phenotype in the chick embryo.

In order to study the influence of ethanol on GABAergic neuronal expression in the chick embryonic brain, activity of glutamate decarboxylase (GAD; a GABAergic neuronal marker) was examined in whole brain derived from embryonic chick following the in ovo administration of ethanol. GAD activity was already detectable by 3 days of embryogenesis and exhibited a logarithmic increase up to embryonic day 16 (E16). By E18, GAD activity began to plateau. Administration of ethanol (10 mg/50 microliters/day) to embryos in ovo on days E1-E3, resulted in increased GAD activity in whole brain when embryos were sacrificed at E8. However, when embryos were administered ethanol on days E4-E7, activity of GAD was unchanged as compared with controls. Similarly, when embryos were administered ethanol chronically from E1-E7 no significant effect was observed in GAD activity. The increase observed in GAD activity after exposure to ethanol during a period of active neuronal proliferation (E1-E3) supports our view that E1-E3 is the critical period of sensitivity to ethanol with respect to the development of neuronal phenotypes during embryogenesis. Furthermore, the absence of any significant effect of ethanol administered from E1 to E7 on GAD activity suggests the existence of a compensatory response to ethanol insult.

Animals↗
PubMed1991

Muscle-derived factors induce proliferation and astrocytic phenotypic expression in C-6 glial cells.

Previous studies from our laboratory have shown that C-6 glial cells in culture express astrocytic phenotypes with increasing cell passage. Early passage glial cells have been shown to respond to various factors by expressing either astrocytic or oligodendrocytic phenotype. In view of the numerous reports on the trophic effects of muscle-derived factors on spinal cord neurons, we examined the effect of muscle-derived factors on C-6 glial cells from both early and late passages. Soluble factors were obtained from either leg (LME) or breast (BME) muscle from 15-day-old chick embryos by homogenization followed by high velocity centrifugation. Both LME- and BME-derived factors triggered the proliferation and differentiation of early passage glial cells as assessed by cell number and glutamine synthetase activity, respectively. Late passage glial cells responded to muscle-derived factors with enhanced proliferation and only with a slight increase in glutamine synthetase activity. These findings indicate that glial cells of both early and late passages respond to signals produced by muscle cells.

Animals↗
PubMed1991

Platelet-activating factor increases glutamine synthetase activity in early and late passage C-6 glioma cells.

Previous studies from this laboratory have shown that C-6 rat glioma cells (2B clone) exhibit specific phenotypic characteristics depending on passage in culture and that these populations respond differentially to addition of various exogenous compounds to the medium. Early passage (less than 25) C-6 glial cells express low glutamine synthetase activity (a marker for astrocytes) and with increasing cell passage (greater than 70) C-6 glial cells express more astrocytic properties with respect to both glutamine synthetase (GS) and morphology. In this study, cells from both early (glioblastic) and late (astrocytic) passage were examined for their response to the phospholipid, platelet-activating factor (PAF). We found that PAF increased GS activity in early passage (glioblastic) cells and more importantly it increased GS activity in late passage cells, already committed to the astrocytic phenotype. Furthermore, cells from both passages failed to respond to addition of lyso-PAF, the non-biologically active analog of PAF, to the medium. By following the uptake of 3H-PAF into cells, we observed that greater than 90% of the phospholipid was taken into the cells within the first hour of incubation. We compared the PAF effects with that of dibutyryl cyclic AMP (dBcAMP) and RO20-1724, a phosphodiesterase inhibitor. Cells from the early passage responded to both dBcAMP and RO20-1724 treatments with a significant increase in GS activity whereas cells from the late passage showed no significant change, confirming earlier reports from this laboratory. These findings indicate that the response of C-6 glioma cells to PAF (at least in the late passage) is not mediated via cyclic AMP. We suggest that in early passage cells PAF promotes expression of the astrocytic phenotype and in late passage cells PAF mediates a gliosis-type response.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1991

Correlation between morphological and biochemical effects of ethanol on neuroblast-enriched cultures derived from three-day-old chick embryos.

We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth. In this study we examined the direct effects of ethanol exposure on developing neuroblasts in culture. Neuroblast-enriched cultures derived from 3-day-old whole chick embryos were grown in the presence of ethanol at doses ranging from 12.5 to 50 mM from culture day 3-14. Cholinergic and GABAergic phenotypic expression were both significantly reduced following ethanol exposure as assessed by the activities of choline acetyltransferase and glutamate decarboxylase, respectively. Morphometric analysis of the growth patterns showed significant differences between control and ethanol-treated cultures. Control cultures exhibited the characteristic pattern of growth consisting of neuronal aggregation with neuritic arborization, i.e., neuritic bundles and fasciculation. Cultures grown in ethanol from culture day 3 consisted of aggregates that measured significantly greater in size than those observed in control cultures. In addition, in ethanol-treated cultures, the primary pattern of neuritic bundles was replaced by a complex network of individual neurites radiating from the central aggregate, forming a defined "neuritic field." Morphometric analysis revealed that both neurite number and neurite length were significantly reduced in ethanol-treated cultures. The biochemical data confirm earlier reports from this laboratory suggesting that ethanol exposure during early embryogenesis alters the normal neuronal pattern of phenotypic expression. In addition, we have presented evidence in this study that ethanol alters the morphological growth patterns of developing neurons. Although ethanol does not alter the ability of these cells to aggregate, there is a significant alteration in neuritic outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Survival and proliferation in developing neuroblasts in cultures derived from embryos treated with ethanol during early neuroembryogenesis: effects attenuated by somatostatin.

Ethanol exerts profound effects both in ovo and in culture on neuronal phenotypic expression. In order to define more directly the neuronal growth parameters sensitive to the neurotoxic effects of ethanol, embryos were exposed to ethanol (10 mg/50 microliters/day) or saline (control) at embryonic days 1 and 2; neuron-enriched cultures prepared from these treated embryos at 3 days of embryonic age. Cultures from both groups were labeled with [3H]thymidine and assessed for effects on neuronal survival and proliferation in culture. Treatment of embryos with ethanol in ovo resulted in a marked enhancement in normal neuronal death in culture with no significant effect on proliferation. Whereas somatostatin (SRIF) had no effect on normally occurring neuronal death in control cultures, addition of SRIF (100 nM) to the culture medium attenuated the ethanol-induced neuronal cell death. Videometric analysis revealed that pretreatment of embryos with ethanol resulted in formation of more and larger aggregates as compared with controls, an effect that was augmented by addition of SRIF to the medium. The most profound effect of ethanol on growth patterns was observed in neurite outgrowth showing a marked reduction in both neurite number and length in embryos pretreated with ethanol. SRIF enhanced neurite outgrowth (neurite number and length) in cultures derived from ethanol-treated embryos. These results suggest that the ethanol-induced deficits in neuronal survival and morphology are reversible. We conclude that SRIF may enhance neuronal survival by promoting neuritic outgrowth, thus establishing the essential target cell contacts necessary for cell survival.

Animals↗
PubMed1991

Growth factors attenuate the cholinotoxic effects of ethanol during early neuroembryogenesis in the chick embryo.

The interaction between growth factors and ethanol on cholinergic neuronal expression was studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase activity as a cholinergic marker. As we have previously reported (Brodie & Vernadakis, Dev. Brain Res. 56: 223-228, 1990; Kentroti and Vernadakis, Dev. Brain Res. 56: 205-210, 1990), ethanol administration in ovo at embryonic days 1-3 produced a 30% decrease in choline acetyltransferase activity. Nerve growth factor and epidermal growth factor administration alone, at embryonic days 1-3, produced a slight increase in choline acetyltransferase activity of both brain and spinal cord when examined at embryonic day 8. Concomitant administration of either nerve growth factor or epidermal growth factor with ethanol eliminated the decrease in choline acetyltransferase activity produced by ethanol. Moreover, administration of either nerve growth factor or epidermal growth factor at embryonic days 4-7 to embryos pretreated with ethanol at days 1-3 raised choline acetyltransferase activity to a level similar to that observed in controls. Thus the growth factors reversed the ethanol-induced cholinergic insult and restored the cholinergic population to normal. These findings provide evidence of a possible role of NGF and EGF in interfering with the neurotoxic effects of ethanol during embryogenesis.

Animals↗