Nitric oxide regulates cell survival in purified cultures of avian retinal neurons: involvement of multiple transduction pathways

Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S-nitroso-acetyl-d-l-penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8-day-old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re-fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re-feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration- and time-dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or LY83583, and mimicked by 8-bromo cyclic guanosine 5'-phosphate (8Br-cGMP) (GMP) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol-3 kinase, extracellular-regulated kinases, calmodulin-dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore considered an important mediator of apoptosis and necrosis, as well as boc-aspartyl (OMe) fluoromethylketone (BAF), a caspase inhibitor, also blocked cell death induced by re-feeding the cultures. These findings demonstrate that NO inhibits apoptosis of retinal neurons in a cGMP/protein kinase G (PKG)-dependent way, and strengthens the notion that NO plays an important role during CNS development.     

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ～30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [³H]-d-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.     

Amitriptyline inhibits neurite outgrowth in chick cerebral neurons: A possible mechanism

Previous studies showed that amitriptyline (AMI), a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants and inhibited adenylyl cyclase activity in cerebral membrane preparations. In the present study, we have investigated the possibility that AMI may have additional effects on cellular metabolism and signal transduction that underlie AMI-mediated inhibition of neurite outgrowth. In vitro AMI inhibited phospholipase C in a dose- and GTP-dependent manner in membranes from 8-day-old chick forebrain. Brain homogenates from 8-day-old chick embryos, treated in vivo for 6 days with AMI (20 μg/g/day), showed significant reductions in (1) phosphorylation of two polypeptides (49 and 105 kD), and (2) levels of three polypeptides (43, 53, and 92 kD). Western blots showed that the 43- and 53-kD polypeptides corresponded to actin and tubulin, respectively. Diolein and dilinolein, potent activators of protein kinase C, stimulated neurite outgrowth and reversed the inhibitory effects of AMI. Sphingosine, a protein kinase C inhibitor, significantly inhibited neurite outgrowth and eliminated the stimulatory effects of diolein and dilinolein on neurite outgrowth. These data suggest that AMI-mediated inhibition of neurite outgrowth involves multiple effects on cellular metabolism and signal transduction. A hypothesis consistent with our data is that AMI interferes in some manner with the action of G proteins in the signal transduction cascade. © 1993 John Wiley & Sons, Inc.     

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.     

Characterization of Glutamate-Induced Formation of N-Acylphosphatidylethanolamine and N-Acylethanolamine in Cultured Neocortical Neurons

Abstract: Glutamate-induced formation of N-acylethanolamine (NAE) and N-acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [¹⁴C]ethanolamine. The formation of these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate-induced formation was seen in 2-day-old cultures, whereas glutamate induced a pronounced formation in 6-day-old cultures. The calcium ionophore A23187 (2 µM) stimulated, within 2 h, formation of NAPE in 2-day-old cultures (fourfold) as well as in 6-day-old cultures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA-selective receptor antagonists d -(?)-2-amino-5-phosphonovalerate and N-(1-(2-thienyl)-cyclohexyl)piperidine and the lack of effect of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In 6-day-old cultures, exposure to NMDA (100 µM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40–50% neuronal death, as measured by a decrease in cellular formazan formation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay]. The increase in NAPE and NAE could be detected earlier than the neuronal death. Neither cyclic AMP, cyclic GMP, nitric oxide, protein kinase C, nor peroxidation appears to be involved in the formation of NAPE and NAE, as assessed by the use of different pharmacological agents. Exposure to 5 mM NaN₃ for 8 h resulted in a >80% decrease in the cellular MTT staining and a pronounced linear increase in the formation of NAE and NAPE (reaching 25–30% of total labeling). [¹⁴C]Anandamide was also formed in [¹⁴C]arachidonic acid-labeled neurons exposed to NaN₃. No NAPE formation was detected in A23187-stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate-induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

Adenosine Regulates the Survival of Avian Retinal Neurons and Photoreceptors in Culture

Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were incubated for 3 days and refed with fresh medium, a large proportion of neurons died in the subsequent 3 days of culture. This cell death was prevented by preincubation of cultures for at least 24h with adenosine plus the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine uptake blocker nitrobenzylthioinosine (NBI), the adenosine A_2A receptor agonist 2-[4-(2-carboxyethyl) phenethylamino]-5-N-ethylcarboxamidoadenosine (CGS21680), or the permeant cyclic AMP analog 8-bromo cyclic AMP, but not the A₁ receptor agonist cyclohexyladenosine (CHA). Adenosine deaminase induced cell death when added to culture medium, and this effect was prevented by EHNA. Cell death was not observed when the medium was replaced by a conditioned medium from sister cultures. The data strongly suggest that adenosine regulates the survival of developing retinal neurons by a long-term activation of A_2A receptors and the increase of cyclic AMP levels.     

Cell Kinetic Studies of Chick Brain Cells In Culture: an Autoradiographic Study With [3H]- and [14C]-Thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [³H]- and/or [¹⁴C]-thymidine. the dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. the labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [³H]- and [¹⁴C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. the cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). the results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

Survival of Chick Embryonic Sensory Neurons in Culture Is Supported by Phorbol Esters

Sensory neurons of the chick embryo are supported in culture by several neurotrophic factors, including the phorbol esters. Because phorbol esters are known to activate one of the second messengers, namely, protein kinase C, it was of interest to see if the neurotrophic action of phorbol 12,13-dibutyrate (PDB) was related to the activation of protein kinase C in sensory neurons. Sensory neurons were obtained from dorsal root ganglia of 10-day-old chick embryos and maintained in a serum-free medium for several days to quantify survival and analyze protein kinase C activity. PDB (30 nM) supported the survival of approximately 50% of the total number of neurons plated. This value was comparable to that supported by nerve growth factor (NGF; 40 ng/ml). If PDB and NGF were added together, there was no additive effect on the survival. The protein kinase C activity of the particulate and cytosolic fractions of sensory neurons supported by NGF for 3 days was 1.26 +/- 0.1 and 2.9 +/- 0.32 pmol/min/mg of protein, respectively. In contrast, neurons supported by PDB showed an approximately 500% increase in enzyme activity in their particulate fraction. The enzyme activity of the cytosolic fraction was decreased by approximately 40%. If NGF-supported neurons were treated with PDB (30 nM) for 15 min, protein kinase C activity increased greater than 400% in the particulate fraction, whereas an approximately 50% decrease was observed in the cytosolic fraction. The protein kinase C value, expressed as a ratio of the activities in the particulate to cytosol fractions, showed large increases after phorbol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic differentiation of red and white skeletal muscle in vivo and in monolayer culture

Cytochrome oxidase, succinate oxidase and lactate dehydrogenase were compared in: (a) leg and breast muscle from 11-19-day-old chick embryos; and (b) 2, 6, 10 and 14-day-old primary cell cultures established from myoblasts of embryonic leg and breast muscle. Cytochrome oxidase, succinate oxidase and lactate dehydrogenase activities were higher (48.8, 65.4, 277.6%, respectively) in leg muscle after 19 days in ovo. Cytochrome and succinate oxidase activities were higher (111.3, 48.1%, respectively) in leg muscle cell cultures after 14 days in vitro. The data represent evidence for intrinsic developmental patterns for certain enzymes.     

Angiotensin II-induced increase of T-type Ca2+ current and decrease of L-type Ca2+ current in heart cells

The effect of angiotensin II (Ang II) on the T- and L-type calcium currents (I(Ca)) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10(-7)M) increased the T-type calcium current and decreased the L-type I(Ca). The effect of Ang II on both types of currents was blocked by the AT1 peptidic antagonist, [Sar1, Ala8] Ang II (2 x 10(-7)M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca2+ currents differently, and this effect seems to be mediated by the PKC pathway.     

Effects of phenytoin on acetylcholinesterase activity and cell protein in cultured chick embryonic skeletal muscle

Cultured pectoral muscle from 11-day-old chick embryos was treated for 48 h with phenytoin (diphenylhydantoin, DPH) in concentrations ranging from 15 to 270 microgram/ml on days 7-9 in vitro. Acetylcholinesterase (AChE, EC 3.1.1.7), creatine phosphokinase (CPK, EC 2.7.3.2), and lactic dehydrogenase (LDH, EC 1.1.1.27) activities, [3H]leucine incorporation into protein, and total protein of the cultures decreased in a dose-related manner with DPH concentrations of 30 microgram/ml and greater. Total AChE activity and AChE activity released into the medium were specifically decreased with 15 microgram DPH per millilitre. In cultures treated chronically with 15 microgram DPH per millilitre on days 5-13 in vitro, total AChE activity and AChE activity released into the medium were 66.0 +/- 13.2 and 64.7 +/- 11.8% of untreated controls, respectively, but cellular AChE activity, cell protein, and [3H]leucine incorporation into protein were unaffected. The results indicate that DPH specifically decreases the total net synthesis of AChE activity by a direct action on cultured chick embryo muscle.     

Complexity of nuclear and polysomal RNAs in growing Dictyostelium discoideum cells

The proliferative activity of undifferentiated brain cells from either 5- or 7-day-old chick embryos has been investigated by labeling the cells with a 24-hr pulse label of [¹⁴C]- or [³H]-thymidine during the early stages (0 to 8 days) of culture. As soon as the neurons and the glial cells could be distinguished (after 4, 7, or 14 days of culture), the cultures were prepared and submitted to the activated autoradiographic method. In some experiments a continuous labeling was applied up to 2 weeks. During the first 48 hr of culture, and for both embryonic ages studied, nearly all neuronal precursors were able to proliferate. After 4 days in culture for the 7-day-old embryo and 7 days in culture for the 5-day-old embryo most of the neuronal cells stopped dividing. These two culture periods correspond to the stage of the embryonic life when the end of the mitotic activity of neuroblasts occurs in vivo. Thus, the proliferation and development in culture of most neuroblasts was found to parallel the in vivo evolution of these cells. Some neuroblasts, however, continued to multiply in vitro for a longer period of time. The astroblasts precursors were found to multiply actively from the 3rd day on, or immediately from time zero, for the 5- and 7-day-old chick embryos, respectively. These observations seem to indicate that the astroblast precursors are in a latent stage until they have reached Day 7. Thereafter, they proliferate actively during the first week of culture and therefore remain in an embryonic stage during this culture period. This fact corresponds also to the in vivo situation, where the glial cell precursors multiply actively around the same time period.     

Adenosine A(2a) receptor-mediated inhibition of rod opsin mRNA expression in tiger salamander

The neuromodulator adenosine mediates dark-adaptive changes in retinal photoreceptors through A(2a) receptors. In cold-blooded vertebrates, opsin mRNA expression is lower at night than during the day. In the present study, we tested whether adenosine could inhibit opsin mRNA expression in cultured rod cells and if endogenous adenosine acts to suppress opsin mRNA in the intact retina at night. Semi-quantitative in situ hybridization showed that treatment with 100 nm of the A(2a)/A(2b) agonist N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) reduced opsin mRNA 41% in cultured rod cells. The effect of DPMA was blocked by 10 microm of the A(2a) antagonist 8-(3-chlorostyryl)caffeine (CSC) but not by 10 microm of the A(2b) antagonist alloxazine. One micromolar adenosine alone had no effect on opsin mRNA. However, in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA), 1 microm adenosine reduced opsin mRNA 61%. EHNA alone reduced opsin mRNA by 26%. Consistent with an A(2a) receptor mechanism, 100 nm forskolin (adenylate cyclase agonist) decreased opsin mRNA 34%. Finally, northern blots showed that intravitreal injection of 10 microm CSC at night increased opsin I mRNA 38%. Thus, endogenous adenosine suppresses rod opsin I mRNA expression at night; in vitro results indicate this reduction occurs through A(2a)-like receptor binding and stimulation of adenylate cyclase activity.     

2’-Deoxyadenosine causes cell death in embryonic chicken sympathetic ganglia and brain

Previous work has shown that nucleosides produce apoptosis in sympathetic ganglion (SG) cells in vitro. The present study examined the effects of nucleosides on the development of the chick embryo in vivo with special attention to the SG and the optic tectum of the central nervous system. In the presence of an adenosine deaminase inhibitor, adenosine and 2'-deoxyadenosine (2'-dAdo) produced different toxicity patterns: both adenosine and 2'-dAdo were toxic to E3 embryos, but only 2'-dAdo was toxic at later stages (E6 1/2, E11). Dosage experiments on E6 1/2 embryos showed that adenosine was less toxic than 2'-dAdo and that 2'-dAdo in sublethal doses was teratogenic. We also examined the effects of 2'-dAdo on embryonic chicken SG and optic tectum in vivo to determine whether sublethal doses of 2'-dAdo produced cell death in these centers on E6 1/2 and 10. In the E6 1/2 SG, 2'-dAdo produced significant neuron loss (83%) and a decrease in SG volume (65%); however, at E10, there was only minor cell loss (7%) and no significant change in SG volume. In the optic tectum at E6 1/2, cell loss was confined mainly to the tectal ventricular zone, but there was little sign of cell loss in this organ at E10. Since cell production is vigorous in the SG and optic tectum at E6 1/2 but relatively low at E10, 2'-dAdo appears to work by stopping cell proliferation. The ineffectiveness of 2'-dAdo at E10 may result from the lethality of 2'-dAdo to the embryo at low concentrations (30 microM) in vivo, well below the apoptosis-inducing concentrations employed in vitro (100-300 microM). These data extend previous findings showing that purine and pyrimidine metabolism plays an important role in development.     

Effects of N-LAAM on [3H]etorphine binding in neuronal-enriched cell cultures

Stereospecific [3H]etorphine binding sites are present in neuronal-enriched cell cultures dissociated from 7-day-old chick embryonic brain. Moreover, binding was regulated by both ions and GTP in a manner similar to that of in vivo brain tissue. When cultures were exposed to N-LAAM (10(-6) M) from day 6 to day 7 or 8 and assayed for binding at day 8, Bmax was decreased and KD was increased. These findings support our view that primary neuronal cultures are a suitable model with which to study interactions of drugs with opiate receptors.