2'-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons.

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.

Cloning and developmental expression of a membrane-type matrix metalloproteinase from chicken.

We have cloned a novel matrix metalloproteinase (MMP) from cultured chicken embryo fibroblasts. The cDNA-derived protein sequence contains 608 amino acids including a C-terminal hydrophobic transmembrane domain of 24 amino acids and a cytoplasmic domain of 20 amino acids. This chicken MMP is 72% similar to a recently described membrane-type MMP (MT-MMP) from human placenta (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65). Accordingly, we name this novel MMP chicken MT-MMP. As shown by Northern blotting, two MT-MMP mRNAs of 6 and 10 kilobases are constitutively expressed but only modestly regulated by growth factors and cytokines in cultured chicken embryo fibroblasts. Both mRNAs are abundant in the head and body of 8- and 9-day-old chicken embryos. As shown by in situ mRNA hybridization, MT-MMP is expressed in embryonic neural tube, spinal ganglia, and respiratory epithelium, as well as in developing cartilage and muscle. Using reverse transcription-polymerase chain reaction, we have found MT-MMP mRNA in 2-day-old chicken embryos and extraembryonic membranes. In addition, a strong correlation was observed between the mRNA expression of MT-MMP and 72-kDa type IV collagenase. Collectively, the early MT-MMP mRNA expression and its co-localization in several tissues with 72-kDa type IV collagenase mRNA suggest that the MT-MMP plays an important role in early development.

Plasma membrane calcium ATPase in synaptic terminals of chick Edinger-Westphal neurons.

The plasma membrane calcium ATPase pump (PMCA) is one of two major mechanisms known to be involved in extruding calcium from cells. The monoclonal antibody 5F10 was used to examine the distribution of PMCA in chick Edinger-Westphal neurons, a population of cholinergic preganglionic neurons whose cells bodies reside in the Edinger-Westphal nucleus in the brainstem and whose axons form synaptic terminals on parasympathetic neurons in the ciliary ganglion. Definitive PMCA immunoreactivity was undetectable in Edinger-Westphal cell bodies in the brainstem. In contrast, immunoreactivity for PMCA was robust in ciliary ganglia and resembled patterns of immunoreactivity for the synaptic vesicle antigen SV-2, suggesting that PMCA is expressed in Edinger-Westphal synaptic terminals. Moreover, PMCA immunoreactivity co-localized with immunoreactivity for enkephalin and substance P, two neuropeptides known to be expressed in Edinger-Westphal synaptic terminals. Fine structure studies revealed that PMCA immunoreactivity is associated with synaptic vesicles rather than the plasma membrane in Edinger-Westphal terminals. In immunodot assays, synaptic vesicles purified from Torpedo electric organ are also immunoreactive for PMCA as well as SV-2. Torpedo vesicles are negative for the sarcoplasmic/ endoplasmic reticulum ATPase, suggesting that the observed PMCA immunoreactivity is not associated with smooth endoplasmic reticulum. Immunoblot analysis confirmed that 5F10 recognizes a protein with the correct molecular mass for PMCA in tissue homogenates of chick cerebellum, chick ciliary ganglia, and Torpedo synaptic vesicles. These findings describe a previously unrecognized location for PMCA in the membranes of cholinergic synaptic vesicles. Relevance to previous data and possible functions are discussed.

Agrin mRNA expression in the developing chick Edinger-Westphal nucleus.

Agrin is a large extracellular matrix protein that directs the accumulation of acetylcholine receptors at the neuromuscular junction. Recent evidence suggests that agrin may be involved in organizing synapses in the visual system as well. Focussing on the pathway that controls accommodation and pupilloconstriction, this study examined the temporal pattern of agrin expression with reference to the organization of cholinergic synapses between embryonic chick Edinger-Westphal and ciliary ganglion neurons. In situ hybridization with an S35-labeled agrin cRNA probe was used to characterize agrin expression in the Edinger-Westphal nucleus during development. Agrin mRNA was detected in the Edinger-Westphal nucleus at all time points studied, from embryonic day 7 (E7, Hamburger and Hamilton stage 31) through newly hatched chicks. Throughout this period, agrin mRNA expression in Edinger-Westphal neurons was lower than in nearby oculomotor and trochlear neurons, suggesting that cells projecting to neuronal targets may require less agrin than those projecting to muscle. Agrin mRNA expression in the Edinger-Westphal nucleus at E7, E8, E9, and E10 was significantly higher than at E12. The early appearance of agrin mRNA coincides with the period during which acetylcholine receptors are being organized on ciliary ganglion neurons, consistent with the possibility that agrin contributes to neuron-neuron synapse formation in this pathway.

Development of parvalbumin immunoreactivity in the chick Edinger Westphal nucleus.

To determine when the calcium-binding protein parvalbumin appears during development, neurons in the chick Edinger Westphal nucleus were examined for parvalbumin immunoreactivity at a variety of embryonic stages. Parvalbumin immunoreactivity appeared on embryonic day 14 (E14, Hamburger and Hamilton stage 40) in predominantly lateral Edinger Westphal neurons. Cytochrome oxidase activity within the nucleus was examined throughout development, as an indicator of physiological activity, and expression of cytochrome oxidase was compared with that of parvalbumin. Cytochrome oxidase activity was found to be uniformly high in all parts of the Edinger Westphal nucleus throughout development. Either the Edinger Westphal nucleus in physiologically active quite early in its development or other energy demands mask the correlation of cytochrome oxidase with electrical activity. Cytochrome oxidase was expressed well before parvalbumin immunoreactivity appeared. Voltage-activated calcium currents were characterized in E12 Edinger Westphal neurons. In both amplitude and composition, E12 calcium currents resemble those of E16 neurons, excluding the possibility that calcium currents appear de novo during or just prior to the appearance of parvalbumin. Both cytochrome oxidase activity and calcium currents are observed in Edinger Westphal neurons well before the appearance of parvalbumin during development. These findings do not exclude the possibility that physiological activity affects the expression of parvalbumin since other factors such as changing patterns of synaptic activity or the appearance of calcium conducting NMDA receptors have yet to be examined. However, they raise the possibility that additional factors such as an intrinsic developmental program or a change in the neuron's basal intracellular calcium requirements may also be involved.

Multiple subtypes of voltage-gated calcium currents in the Edinger-Westphal nucleus.

Avian Edinger-Westphal (EW) neurons provide a unique opportunity to compare electrophysiologically the membranes of cell bodies and terminals in the same population of neurons. Axons that originate from neurons in the lateral region of the EW nucleus form a morphologically distinct presynaptic terminal, known as a calyx, on ciliary ganglion neurons. Several studies have shown that calyciform terminals in the ciliary ganglion exhibit predominantly N-type, high-voltage-activated (HVA) calcium channels. The goal of this study was to characterize and compare the calcium currents expressed in EW cell somas with those reported in the terminals. Whole-cell patch-clamp techniques were used to record from cell bodies in the lateral EW nucleus in slice preparations. Slices were obtained from embryonic day 16 chicks, matching the age of the embryos in which calyces were studied. Recordings were localized to the lateral region of the EW nucleus using Lucifer yellow fills. Voltage-step commands from -70 to 0 mV produced calcium currents with both a sustained and an inactivating component. Depolarization steps to 0 mV from a holding potential of -40 mV eliminated the inactivating component. These recordings suggested the presence of both LVA and HVA calcium currents. Application of 0.1 mM NiCl2 produced a reversible decrease in the amplitude of the whole-cell calcium current, preferentially affecting the inactivating component. The Ni2+(-)sensitive current activated and inactivated rapidly in a voltage-dependent manner. Treatment with 0.1 mM cadmium chloride caused a reversible reduction in the amplitude of the calcium current.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic vesicle proteins and target-dependent morphogenesis of calyx-like contacts in vitro.

Chick Edinger-Westphal neurons form large, calyciform synaptic terminals, also known as calyces, on a subpopulation of ciliary ganglion neurons in vivo. This distinctive morphology is mimicked by contacts formed between Edinger-Westphal and ciliary ganglion neurons in culture. The work reported here finds that calyx-like contacts in culture are immunoreactive for the two vesicle proteins SV-2 and synaptotagmin, supporting the hypothesis that these contacts are synaptic terminals developing in vitro. When Edinger-Westphal neurons are cultured with ciliary ganglion neurons, calyx-like contacts are three times more frequent than when Edinger-Westphal neurons are cultured inappropriately with sympathetic neurons. Furthermore, calyx-like contacts are rare in cultures of ciliary ganglion neurons alone, even though ciliary ganglion neurons are known to functionally innervate themselves in culture. These results suggest that calyx morphogenesis is favored when Edinger-Westphal neurons interact specifically with ciliary ganglion neurons and discouraged when they interact with inappropriate neurons. That this specificity is expressed in culture, in the absence of normal tissue topography and patterned activity, adds to the evidence that direct surface interactions between target cells and growing neurites are involved in the morphogenesis of synaptic terminals in vivo.

Characterization and development of glial and other non-neuronal cells in chick Edinger Westphal cultures.

Cultures of dissociated Edinger Westphal nuclei, dissected from embryonic chick brainstems, were screened immunohistochemically for a variety of non-neuronal cell markers. In young cultures, small clusters of cells were stained by the oligodendrocyte-specific antibodies 04 and 01. In older cultures, larger groups of cells were 04 and 01 positive, sheets of myelin-like membrane were elaborated, and immunoreactivity for proteolipid protein appeared. This sequence resembles that observed in well-characterized rodent brain cultures and suggests that oligodendrocytes in chick Edinger Westphal cultures differentiate in a pattern similar to rodent oligodendrocytes in culture. Variable numbers of cells were immunoreactive for glial fibrillary acidic protein. Many vimentin positive cells were observed, some of which morphologically resembled flat astrocytes. Together with the widespread presence of vimentin, large patches of fibronectin-like immunoreactivity suggested the presence of fibroblasts and/or endothelial cells. An anti-thymocyte polyclonal antibody stained a subset of cobblestone-shaped cells, possibly endothelial cells, in both Edinger Westphal cultures and control cultures of skin fibroblasts. Staining for smooth muscle myosin was detected in several patches of cells, tentatively identifying them as pericytes or smooth muscle cells. In conclusion, Edinger Westphal cultures contain a diverse and varying population of non-neuronal cells loosely organized in large, overlapping islands of cell types and including oligodendrocytes, astrocytes, possibly fibroblasts, endothelial cells, pericytes and/or smooth muscle cells.

Calcium-binding proteins in the chick Edinger Westphal nucleus.

It has been suggested that the calcium-binding proteins, parvalbumin and calbindin-D28K, are involved in the control of intracellular calcium levels but their exact functions are unknown. Immunoreactivity for parvalbumin has been associated with rapidly firing cells, while calbindin has been implicated in protecting neurons from excitotoxicity. Since the chick Edinger Westphal nucleus contains two populations of neurons with different firing patterns, parvalbumin and calbindin immunoreactivities were examined in the Edinger Westphal nuclei of posthatch chicks to determine whether a particular subpopulation of neurons is associated with either protein. Moderate levels of parvalbumin immunoreactivity were found consistently associated with repetitively firing neurons in the lateral Edinger Westphal nucleus. In contrast, medial neurons expressed much lower levels of parvalbumin immunoreactivity. Many medial neurons were negative for parvalbumin although occasionally a few medial neurons stained as strongly as lateral neurons. Definitive calbindin immunoreactivity was absent from Edinger Westphal nuclei despite robust staining of cells in other parts of the brainstem and in control sections of cerebellum.

Repetitive firing properties in subpopulations of the chick Edinger Westphal nucleus.

The avian Edinger Westphal nucleus, through the ciliary ganglion, controls accommodation, iris constriction, and blood flow through the choroid. In live brainstem slices, the nucleus is easily identifiable as an olive-shaped cluster of neurons dorsal to the oculomotor nerve and nucleus. Intracellular recordings from neurons in the nucleus identified two classes of responses to sustained (300 to 500 ms) injections of depolarizing current. One set of cells fired action potentials for the duration of the pulse while a second set of cells fired action potentials only transiently, during the first 50 to 100 ms, after which they remained silent regardless of the size of the depolarization. Intracellular recordings followed by injections of the fluorescent dye lucifer yellow revealed that repetitively firing cells were located in the lateral half of the nucleus while non-repetitively or transiently firing cells were located in the medial half. These locations correspond to different Edinger Westphal subdivisions which have distinct inputs and target populations. The varying firing patterns are discussed with reference to the known functions of the subdivisions in which they occur. Replacement of calcium by magnesium in the extracellular medium had no effect on the number of action potentials fired by non-repetitively firing cells, suggesting that a calcium-activated potassium current is not responsible for suppressing repetitive firing in these cells. In contrast, in repetitively firing cells removal of extracellular calcium increased the frequency of action potential discharge and decreased the amplitude of afterhyperpolarizations following single action potentials. Addition of cadmium to the bath medium had similar effects.(ABSTRACT TRUNCATED AT 250 WORDS)

A Na+-activated K+ current in cultured brain stem neurones from chicks.

1. Patch-clamp techniques were used to study the properties of a Na+-activated K+ current (IK(Na) in neurones cultured from embryonic chick brain stem. 2. With whole-cell clamp, a depolarizing voltage command evoked an inward current that was followed by an outward current with two components, the first transient, the second sustained. 3. Tetrodotoxin (TTX, 1 microM) eliminated the inward current and the transient component of the outward current, without affecting the sustained outward current. In addition, the transient outward current was attenuated when all external Na+ was replaced by Li+, suggesting that it was activated specifically by Na+ entry into the cell. 4. The time course of the transient outward current was obtained by subtracting records obtained in Li+ solution from those obtained in Na+ solution. There was significant overlap between the decay of the inward current and the onset of the transient outward current. 5. When just after the peak of the transient outward current, the membrane was stepped to progressively more hyperpolarized levels, the tail currents associated with the current reversed polarity near the calculated K+ equilibrium potential. 6. 4-Aminopyridine (4-AP, 4 mM) abolished the transient outward current and approximately half of the sustained late current. Tetraethylammonium (TEA, 2 mM) had no effect on the transient current, but reduced the sustained current slightly. 7. Inside-out patches, made in LiCl bathing solutions, contained channels that were activated by exposing the cytoplasmic face of the patch to Na+. Channel activity continued as long as Na+ was present. 8. The single-channel currents reversed at the K+ equilibrium potential, and were associated with a main conductance that depended upon K+ concentration (about 50 pS with [K+]o = 15 mM, [K+]i = 5 mM, and 100 pS when [K+]i was increased to 75 mM). 9. The open probability of the channels increased with increasing cytoplasmic Na+ concentration. At [Na+]i = 150 mM (the maximum concentration tested), channels were open almost continuously. Open probability was considerably less at 50 mM, and still measureable at 20 mM. 10. The magnitude of IK(Na) and its overlap with the inward Na+ current indicate that these channels contribute significantly to the repolarizing phase of the action potential. In addition, the relation between channel activity and Na+ concentration suggests that the channels may make a measurable contribution to membrane conductance at resting intracellular Na+ concentrations.

Formation of calyx-like contacts preferentially on appropriate target neurons in culture.

The availability of culture systems for both Edinger Westphal and ciliary ganglion neurons has made it possible to examine the interactions in culture between two populations of vertebrate neurons that synapse in vivo. In the chick, Edinger Westphal neurons provide the sole presynaptic input to the ciliary ganglion and, through this projection, are responsible for the control of lens curvature (accommodation), iris constriction, and possibly smooth muscle function in the choroid layer of the eye. When embryonic chick Edinger Westphal and ciliary ganglion neurons were combined in culture and stained for enkephalin-like immunoreactivity to visualize Edinger Westphal terminals, stained calyx-like contacts were observed that resemble the calyciform terminals formed between Edinger Westphal processes and ciliary neurons in the ciliary ganglion in vivo. Although stained calyx-like contacts could also be found in Edinger Westphal-alone and ciliary ganglion-alone cultures, many more were observed when the two cell types were cultured together. The increase depended specifically on the ciliary ganglion neurons since substitution of either dorsal root ganglion or sympathetic ganglion neurons for them in the cocultures did not increase the number of calyx-like contacts staining positive for enkephalin over those present in cultures of Edinger Westphal neurons alone. When Edinger Westphal neurons were grown simultaneously with dorsal root and ciliary ganglion neurons, calyx-like contacts with enkephalin-like immunoreactivity were found to terminate preferentially on the latter. These findings suggest that vertebrate neurons can form morphologically specific contacts preferentially on appropriate target cells in culture in the absence of many of the potential cues present in the intact tissue.

Preganglionic neurons from the Edinger-Westphal nucleus: growth and histochemical characterization in cell culture.

The Edinger-Westphal (EW) nucleus, also known as the accessory oculomotor nucleus in the chick, provides the cholinergic preganglionic input to the parasympathetic ciliary ganglion. In addition to acetylcholine, many EW neurons have been shown to contain enkephalin-like and/or substance P-like immunoreactivity. Establishment of EW neurons in culture would make possible study of their interactions with ciliary ganglion neurons in vitro and in addition would provide a valuable system for studying cholinergic/peptidergic neurons of the vertebrate central nervous system. We describe here dissociated cell cultures established from midbrain tissue containing the EW nucleus. In these cultures, 86% of the cells with neuronal morphology were positive for intracellular acetylcholinesterase activity, 54% were positive for enkephalin-like immunoreactivity, and 4% were positive for substance P-like immunoreactivity. The proportions of neurons that scored as labeled were even higher if the number of positive cells was compared to the number of cells in sister cultures immunoreactive for the large neurofilament protein polypeptide. When the cultures were stained simultaneously for acetylcholinesterase activity and enkephalin-like immunoreactivity, 34% of the cells with neuronal morphology were positive for both. In cultures derived from adjacent tissue regions very few cells expressed both activities. These results suggest that the cells expressing both acetylcholinesterase activity and enkephalin-like immunoreactivity in culture are EW neurons. The putative EW neurons survive for weeks in vitro in the absence of their normal target, the ciliary ganglion.

Developmental potential of teratocarcinoma stem cells in utero following aggregation with cleavage-stage mouse embryos.

Embryonal carcinoma cells were aggregated with cleavage stage mouse embryos, cultured briefly, and transferred as morulae to the uteri of pseudopregnant females. When midgestation foetuses were examined, many were morphologically abnormal. The severity of this abnormal development was correlated with the extent of contribution by embryonal carcinoma cells to the foetuses as indicated by GPI (glucose phosphate isomerase) analysis. This was true for all three of the cell lines studied, NG-2, PSA-1, and LT1-2D. The clear correlation between increasingly abnormal development and more extensive participation by the embryonal carcinoma cells was not observed in control experiments in which embryos of different stages of development were aggregated together. The data therefore suggest that the embryonal carcinoma cells studied here are unable to support normal development in the absence of a substantial number of host embryonic cells. It remains unclear whether this is a consequence of the karyotypic abnormalities of the cells tested, or whether it reflects a characteristic limitation in the ability of embryonal carcinoma cells to independently direct normal development. When aggregates were allowed to develop to term and the extent of chimaerism was examined in the live-born animals, it was found to be sporadic and limited. This is consistent with the results indicating that large contributions by embryonal carcinoma cells are not compatible with normal development at midgestation. The chimaerism observed in the live-born animals was comparable in both frequency and in tissue distribution to that generally obtained in other studies using either the aggregation or blastocyst injection techniques.