Chick Embryo Cerebral Hemispheres Research Articles

Effects of Selenoprotein W gene expression by selenium involves regulation of mRNA stability in chicken embryos neurons

Selenium (Se) and Selenoprotein W (SelW) plays a pivotal role in the brain development, function, and degeneration and that SelW expression in the brain may be affected by Se. However, the mechanism which Se regulates the SelW gene expression in neurons remains to be unclear. To investigate the effects of the SelW gene expression and mRNA stability induced by Se, primary cultured chicken embryos neurons derived from 8-day-old chick embryo cerebral hemispheres were treated with 10(-9)-10(-5) mol/l Se as selenite for 3, 6, 12, 24 or 48 h, respectively. The morphology and viability of Neurons was detected. The SelW mRNA expression level and mRNA half-life was examined in Se-treated neurons. The relative low concentrations of Se enhanced the neurite outgrowth, increased the SelW mRNA levels and elevated the mRNA half-life of chick embryo neurons. In contrast, the high concentrations of Se presented neurotoxic to neurons, decreased the SelW mRNA levels and reduced the mRNA half-life of neuronal cells. These results suggest that the alteration of post-transcriptional stabilization of SelW mRNA is an important mechanism of Se-induced the elevation or reduction of the SelW expression level in chick embryo neurons.

Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation.

Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin beta1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.

Differential expression in glial cells derived from chick embryo cerebral hemispheres at an advanced stage of development

Recently, we have characterized glial cultures derived from very early neurogenesis (E3) and found them to consist largely of early glioblastic or astroblastic cells with the capacity to differentiate into astrocytes given sufficient time in culture or with advancing age, i.e., cell passage. This study examines and compares the characteristics of astrocyte-enriched cultures derived from advanced embryonic ages (E15) in the chick embryonic cerebral hemispheres. We report several remarkable findings. 1) Mature astrocytes (GFAP+, vimentin-) appear as early as 5 days in vitro (DIV) in primary culture (P0). 2) Also apparent in primary cultures were extensive populations of neurons (neurofilament+; NF+) growing atop or in close proximity to mature astrocytes. 3) NF+ neurons disappeared after the first cell passage, and GFAP+ astrocytes were greatly diminished within two cell passages thereafter. 3) High concentrations of NGF were expressed, presumably by glial cells, in primary cultures through 14 DIV, declining to a low plateau through 27 DIV and remaining low, but measurable in subsequent cell passages. 4) At later cell passages (> 5) immature phenotypes of these same cell types continued to be expressed in E15CH cultures, i.e., positive staining for GFAP and vimentin and GFAP, GS, and NGF can all be detected on Western blots. We conclude from these findings that 1) mitotic multipotential neural cells are present within cerebral hemispheres even at late stages of development (E15); 2) neuroblasts and astroblasts have a reciprocal relationship requiring the presence of both cell types in order for mature expression of their phenotypes; 3) the NGF profile parallels the appearance and disappearance of neurons in E15 chick embryonic cerebral hemisphere primary cultures, strongly suggesting that this trophic factor may be involved in the mutually beneficial relationship between astrocytes and neurons.

Differential expression in glial cells derived from chick embryo cerebral hemispheres at an advanced stage of development

Differential expression in glial cells derived from chick embryo cerebral hemispheres at an advanced stage of development

THE NANOMELIC MUTATION IN THE AGGRECAN GENE IS EXPRESSED IN CHICK CHONDROCYTES AND NEURONS

We have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK-1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS-2 domain of the cartilage-specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L-reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK-1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene. In the present study, the S103L CSPG is not accumulated or synthesized by embryonic chick CNS tissue or E8CH neuronal cultures derived from nanomelic chick embryo cerebral hemispheres. In contrast, expression of both versican and the HNK-1 CSPG was normal in the mutant embryo CNS. Pulse chase experiments demonstrated the presence of the 380 kDa precursor in normal neurons and the 300 kDa truncated precursor in nanomelic neurons. Northern blot analysis revealed normal-sized mRNA but reduced levels of expression of the S103L CSPG message in nanomelic neurons, while expression of the versican message was comparable in normal and nanomelic neurons. Most conclusively, the point mutation previously identified in nanomelic cartilage mRNA was also identified in nanomelic brain mRNA. Together these results provide evidence that a single aggrecan gene is expressed in both cartilage and CNS tissue leading to the production of identical core proteins which then undergo differential and tissue-specific post-translation processing, resulting in the characteristic tissue-specific proteoglycans. Furthermore, versican and the HNK-1 CSPG, although structurally and chemically similar to the S103L CSPG, are the products of separate genes. Copyright © 1996 ISDN. Published by Elsevier Science Ltd.

Cerebrolysin protects neurons from ischemia-induced loss of microtubule--associated protein 2.

Microtubule — associated protein 2 (MAP2) was studied in cultured neurons from chick embryo hemispheres after neuronal damage caused by cytotoxic ischemia. Cortical cells were pretreated with 0, 10, 20, 40 or 80µ1 Cerebrolysin® per ml media. After 8 days in vitro (DIV), ischemia was induced by a 60 minutes incubation with 1 mM L-glutamate. After this toxic stress, cells were allowed to recover from ischemia for 48 hours in a medium again supplemented with Cerebrolysin® in the appropriate concentration. For immunoblot analysis cortical cells were lysed in 1% SDS and the supernatants were assayed for protein content by the method of Lowry. Proteins were then separated and transferred to nitrocellulose (0.5 A for 2h). Western blots were blocked with low fat milk and incubated with the primary monoclonal antibody (Chemicon) reacting with all forms of MAP2 (Map2a, Map2b, Map2c) for 12–14 hours at 4°C. Incubation with the horseradish peroxidase — conjugated secondary antibody lasted for 2h. For detection we used a nonradioactive ECL (enhanced chemiluminescence) system (Amersham).KeywordsControl CellHorseradish PeroxidaseCortical NeuronImmunoblot AnalysisNeuronal DamageThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differential regulation of phospholipases C and D by phorbol esters and the physiological activators carbachol and glutamate in astrocytes from chicken embryo cerebrum and cerebellum

Primary astrocytic cultures derived from day-15 chick embryo (E15) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-α/β). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP 2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP, hydrolysis in E15CH astrocytes, arguing for contribution of M 1 subtype. The phorbol ester TPA completely inhibited PIP 2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC than that observed with carbachol. We investigated phospholipase D (PLD) activation as an alternate source of diacylglycerol in astrocytes, since the ratio of PLC to PKC activation by carbachol was lower in astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M 3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation in CB astrocytes than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca 2+] i . Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development.

GABA attenuates the neurotoxic effects of ethanol in neuron-enriched cultures from 8-day-old chick embryo cerebral hemispheres

Neuron-enriched cultures were prepared from 8-day-old chick embryo cerebral hemispheres and exposed to ethanol (50 mM), GABA (10 −5 M) and ethanol (50 mM) + GABA (10 −5 M) from day 4 to 8 in culture. At day 8, control, ethanol, GABA and ethanol + GABA-treated cultures were examined morphologically and biochemically. Choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities were used as markers for cholinergic and GABAergic neuronal phenotypic expression, respectively. Control cultures showed more numerous and large neuronal aggregates as well as prominent neuritic bundles. Moreover, cultures treated with GABA depicted even more numerous neuronal aggregates with interconnecting neurites as compared to control. In contrast, ethanol-treated cultures exhibited smaller neuronal aggregates with less prominent neuritic bundles than control. However, cultures treated concomitantly with ethanol + GABA exhibited numerous and larger aggregates than cultures treated with ethanol alone. Neuritic bundles which were highly reduced in ethanol-treated cultures became prominent in the presence of GABA. As previously reported, ethanol alone enhanced ChAT and reduced GAD activities. GABA given alone enhanced the expression of both neuronal phenotypes. When GABA was given concomitantly with ethanol the decline in GAD and the rise in ChAT observed in ethanol-treated cultures was restored by GABA to almost control levels. Thus, ethanol-induced alterations in morphology and neuronal phenotypes were counteracted by the neurontrophic effect of GABA.

Neuroprotective effects of Ginkgo biloba constituents

The neuroprotective effects of Ginkgo biloba extract (EGb 761) and some of its constituents were tested by using the mouse and the rat model of focal cerebral ischemia, the rat model of global cerebral ischemia and primary cultures of neurons obtained from newborn rat hippocampi and chick embryo telencephalic hemispheres. In the models of focal ischemia, 2 days after occlusion of the middle cerebral artery the infarct area on the mouse brain surface and the infarct volume of the rat brain were measured. The infarct area on the mouse brain was dose-dependently (5–20 mg/kg, s.c.) reduced by bilobalide administered 60 min before ischemia. When administered immediately after ischemia, 10 mg/kg bilobalide also diminished the infarct area. The same dose of bilobalide administered to rats 60 min before occluding the middle cerebral artery reduced the cortical and the total infarct volume significantly. Ginkgolide A (50 mg/kg, s.c.) and ginkgolide B (100 mg/kg, s.c.) also had cerebroprotective effects in the mouse model of focal cerebral ischemia, but ginkgolides C and J did not. EGb 761 (2 × 100 mg/kg,i.v.) increased the cerebral blood flow after 10 min of global ischemia in rats but neuroprotection was not demonstrable. Ginkgolide B (1 μM) and bilobalide (10 μM) were shown to protect cultured rat hippocampal neurons against damage caused by glutamate. Bilobalide (0.1 μM) also enhanced the percentage of viable neurons in primary cultures from chick embryo hemispheres when damaged with 1 mM cyanide. The results demonstrate different types of neuroprotective and cerebrovascular effects of the Ginkgo biloba extract and some of its constituents.

Biotransformation of carbon tetrachloride in cultured neurons and astrocytes

The ability of brain neuronal cells to metabolize carbon tetrachloride (CCl 4) has been studied in an attempt to explain earlier observed toxic effects of CCl 4 on these cells. The expression of cytochrome P-450, the glutathione (GSH) content and the activity of glutathione- S-transferase (GST) were measured in cultured neurons and astrocytes from chick embryo cerebral hemispheres. The metabolism of CCl 4 in the neuron and astrocyte cultures was also assessed by determining the formation of: CCl 2 in membrane preparations of these cells. In the membrane fractions of neurons and astrocytes, no measurable levels of cytochrome P-450 were observed. Nevertheless, neurons as well as astrocytes had a capacity for the metabolism of CCl 4. The metabolic capacity of the neurons was significantly greater than that of the astrocytes. The neuron cultures had a higher initial content of GSH and a higher control activity of GST than had the astrocytes. Neither the GSH level nor GST activity were significantly affected in the neuron cultures after exposure to CCl 4. In astrocyte cultures 2 m m CCl 4 slightly depleted the GSH level and significantly induced GST activity. At 3 m m CCl 4, GSH was depleted by 30% and by more than 50% at 4 m m CCl 4. It can be concluded that the metabolic activation of CCl 4 was higher in neurons than in astrocytes. This can explain the earlier observation of CCl 4-induced lipid peroxidation in cultured neurons. Moreover, neuron GSH was not able to protect these cells against CCl 4-induced peroxidative damage. In the astrocytes, on the other hand, GSH and GST appeared to have a role in detoxification of CCl 4.

Protective effects of 5-HT1A receptor agonists against neuronal damage demonstrated in vivo and in vitro.

The aim of the present study was to evaluate the neuroprotective effect of the 5-hydroxytryptamine1A (5-HT1A) agonists, CM 57493 and urapidil, in vivo and in vitro, respectively. In vivo permanent occlusion of the middle cerebral artery (MCA) was performed in male Wistar rats. Forty-eight hours after electrocoagulation of the MCA the infarct volume was determined. Pretreatment of the rat with the 5-HT1A agonist urapidil significantly reduced infarct development. The neuroprotective effect of the agent was restricted to the cortical area; the striatal damage was not influenced. As the stimulation of the 5-HT1A receptor by serotonin is supposed to induce inhibitory, hyperpolarizing effects by opening of a Ca(2+)-independent neuronal K+ ionophore, the efficacy of agonistic drugs directly on the neuron was investigated in vitro. Cyanide-induced cytotoxic hypoxia as well as glutamate-induced excitotoxicity were performed using primary neuronal cell cultures from chick embryo cerebral hemispheres. Treatment with the 5-HT1A agonists urapidil and CM 57493 significantly increased protein content of hypoxic cultures. CM 57493 added to the culture medium (1-10 microM) during and up to 24 h after glutamate exposure ameliorated viability of the neurons. The results demonstrate neuroprotective potency of the 5-HT1A agonists, urapidil and CM 57493, when applied under hypoxic, excitotoxic and ischemic conditions in vivo and in vitro, respectively. Both, presynaptically induced inhibition of glutamate release as well as postsynaptically induced inhibition of neuronal excitability could be discussed as possible mechanisms of action of the 5-HT1A receptor agonism.

Proliferation of neuronal precursor cells from the central nervous system in culture.

Studies over the past ten years have revealed that neuronal precursors from the central nervous system of chick, rat and mouse embryos are able to divide in culture and that their proliferation is enhanced by several nervous tissue extracts as well as by growth factors, hormones and various other molecules. In this article we present an overview of this subject. It has been found that neuronal precursors from chick embryo cerebral hemispheres proliferate in culture during the first week and that those from 6 day-old chick embryos possess the highest proliferative activity. Neuronal precursors from fetal rat cerebral cortex and spinal cord can also proliferate in vitro. The highest proliferative activity was observed between 24 and 48 h. Brain and meningeal extracts have been shown to stimulate the proliferation of chick neuroblasts. Moreover, RNAs, purine nucleotides, purine bases and transferrin present in these extracts are able to reinduce the proliferation of these cells. Other investigations have indicated that several hormones and growth factors stimulate the proliferation of rat and mouse neuronal precursors. Acidic and basic fibroblast growth factors are potent mitogens for these cells. Nerve growth factor, epidermal growth factor and insulin-like growth factor also affect the growth of the neuroblasts. The reported in vitro observations are discussed in relation to the physiological role of these molecules during neuronal proliferation in brain development.

Endothelin-1 exacerbates focal cerebral ischemia without exerting neurotoxic action in vitro

In a model of focal cerebral ischemia in mice, intracisternal injection of 5 and 10 pmol/mouse endothelin-1 significantly increased the infarcted surface area by 15.5% and by 23.5%, respectively. Endothelin-1 (0.01, 1, and 100 nmol/l) added to the primary neuronal cultures of chick embryo cerebral hemispheres for 1 h and 24 h did not influence the viability of the neurons or the protein content of the cultures. When applied simultaneously with 1 mmol/l sodium cyanide for 30 min, endothelin-1 (0.01, 1, and 100 nM) did not modify the hypoxia-induced changes. The results show that exogenously applied endothelin-1 could exacerbate cerebral ischemia, probably due to its vasoconstrictive properties and not to a direct neurotoxic effect.

Ethanol exerts differential effects on high affinity choline uptake in neuron-enriched cultures from 8-day-old chick embryo cerebral hemispheres

Neuronal-enriched cultures were prepared from 8-day-old chick embryo cerebral hemispheres and exposed to ethanol (50 mM) from day 4 to 8 in culture. At day 8, both control and ethanol-treated cultures were processed for [3H]choline uptake in situ. Uptake was performed on cultures containing either Na(+)-plus or Na(+)-free (Li+) HEPES buffer. Total choline uptake as well as Na(+)-dependent and Na(+)-independent choline uptake were calculated. The Km and Vmax were calculated using the Lineweaver-Burke analysis. Our analysis of the data revealed that ethanol-treated cultures exhibited two values for Vmax, one similar to that found in control cultures and one significantly lower than controls. No differences were observed in Km values between control and ethanol-treated cultures. We interpret the low Vmax to represent a population of cholinergic neurons which have been arrested at an immature stage as a result of ethanol insult.

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

Neuronglia 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). Sixday-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.

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 neurotropic signals important for neuronal differentiation.

Vinpocetine increases the neuroprotective effect of adenosine in vitro

Cultured neurons from chick embryo cerebral hemispheres were used to study the neuroprotective activity of vinpocetine and adenosine against hypoxic damage. Cytotoxic hypoxia was induced by adding 1 mM sodium cyanide to the nutrient medium for 30 min. The drugs were present in the nutrient medium 30 min before, and up to 1 day after hypoxia. To characterize the neuroprotective drug effects, the protein content per culture flask and the neuronal viability were determined 3 days after this hypoxic period. Adenosine, but not vinpocetine, was able to protect neurons against hypoxic damage, when added as single drug to the cultures. However, vinpocetine significantly enhanced the neuroprotective effect of adenosine. The results suggest that the neuroprotective infect of vinpocetine is mediated by adenosine.

Chronic opioid treatment attenuates carbachol-mediated polyphosphoinositide hydrolysis in chick embryo neuronal cultures

Opiate binding sites on cultured neurons derived from 6-day-old (E6) chick embryo cerebral hemispheres (CH), shown to be cholinergic by choline acetyltransferase immunostaining, were labeledd with [ 3H]etorphine (μ and δ opiate receptors expression) and [ 3H]morphine (mostly μ). When examined by light microscope autoradiography, opiate receptors were found to be expressed by most neurons and were distributed predominantly on neuronal perikarya. Muscarnic and opiate receptors in E6CH cultured neurons were found to be functionally coupled when the effects of opiate receptor occupancy on the inositol phosphate-linked muscarinic receptors was studied. Carbachol stimulated the release of [ 3H]inositol phosphates (InsP) from cultures preincubated with [ 3H]inositol and LiCl, in a dose-dependent manner, and the functional expression of muscarinic receptors peaked in number at day 7 in culture, declining thereafter. Short-term (<1h) treatment of E6 neuronal cultures with 1 μM opioid peptides such as morphiceptin or d-Ala 2- d-Leu 5-enkephalin (DADLE) did not not inhibit the release of inositol phosphates in response to 1 mM carbachol whereas forskolin, which also activates adenylate cyclase and raises cAMP levels, inhibited InsP release by about 25%. In contrast, long-term (48 h) opioid treatment with either morphiceptin or DADLE (1–10 μM) inhibited the carbachol-stimulated inositol phosphate release by ⩾50%. Prolonged treatment with morphiceptin also inhibited the bradykinin-mediated release of InsP from E6CH cells. In both cases, the inhibition was blocked by the continuous presence of naloxone, suggesting that the inhibition was mediated through opiate receptors. When E6CH cells were depolarized by 30 mM K +, allowing [Ca 2+]i to increase, the inhibition of inositol phosphate formation caused by long-term morphiceptin exposure was partially relieved. This 48 h (chronic) exposure to opioids may involve some depletion of available [Ca 2+]i in cholinergic neurons. We propose that these interactions between the opiate and muscarinic receptor transducing systems may represent the molecular basis of the neuromodulating activity of opioids upon the cholinergic system early in development.

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10(-5)M and 10(-8)M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [3H] and [35S] methionine and [3H]ethanolamine as precursors showed an increased methylation of [3H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [3H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [3H]- and [35S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased 3H- and 35S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [3H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-3H] label after incubation of neurons with [3H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.

Muscle-derived factors enhance cholinergic neuronal expression in the chick embryo—II. In culture studies

The effects of muscle-derived factors on the cholinergic and GABAergic neuronal phenotypes were studied in cultures derived from 3-day-old whole chick embryo (E3WE) consisting of proliferating neuroblasts and 8-day-old chick embryo cerebral hemispheres (E8CH) consisting of differentiated neurons. The effects of limb muscle extract (LME) were examined either when added to the culture medium or to the polylysine coating substratum cultures. We also compared the effects of LME derived from 8- to 15-day-old chick embryos. We found that LME added to the medium not only increased choline acetyltransferase activity, a marker for cholinergic neurons throughout the development of neurons in E3WE or E8CH culture, but also delayed the decline in the activity observed in untreated cultures. The marked increase in choline acetyltransferase activity in E8CH cultures grown on substratum-bound LME as compared to those grown in medium-containing LME, suggests that LME factors may be adhesion-promoting substances stimulating neuronal growth. These findings provide evidence that muscle-derived factors may be important in early cholinergic phenotypic expression and support our previous in ovo studies indicating that target-derived factors in limb muscle extract have general cholinotrophic effect.