Rotational diffusion of acetylcholine receptors on cultured rat myotubes

The rotational mobility of acetylcholine receptors (AChR) in the plasma membrane of living rat myotubes in culture is measured in this study by polarized fluorescence recovery after photobleaching (PFRAP). These AChR are known to exist in two distinct classes, evident by labeling with rhodamine alpha-bungarotoxin; clustered AChR that are aggregated in a pattern of highly concentrated speckles and streaks, with each cluster occupying an area of approximately 1,000 microns 2; and nonclustered AChR that appear as diffuse labeling. PFRAP results reported here show that: (a) most clustered AChR (approximately 86%) are rotationally immobile within a time scale of at least several seconds; and (b) most nonclustered AChR (approximately 76%) are rotationally mobile with characteristic times ranging from less than 50 ms to 0.1 s. External cross-linking with the tetravalent lectin concanavalin A immobilizes many nonclustered AChR. PFRAP experiments in the presence of carbachol or cytochalasin D show that the restraints to rotational motion in clusters are remarkably immune to treatments that disperse clusters or disrupt cytoplasmic actin. The experiments also demonstrate the feasibility of using PFRAP to measure rotational diffusion on selected microscopic areas of living nondeoxygenated cells labeled with standard fluorescence probes over a very wide range of time scales, and they also indicate what technical improvements would make PFRAP even more practicable.     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

Addition of the BMP4 antagonist, noggin, disrupts avian inner ear development

Bone morphogenetic protein 4 (BMP4) is known to regulate dorsoventral patterning, limb bud formation and axis specification in many organisms, including the chicken. In the chick developing inner ear, BMP4 expression becomes localized in two cell clusters at the anterior and posterior edges of the otic epithelium beginning at stage 16/17 and is expressed in presumptive sensory tissue at later stages. This restricted spatiotemporal pattern of expression occurs just prior to the otocyst's transition to a more complex three-dimensional structure. To further analyze the role of BMP4 in avian otic morphogenesis, cells expressing BMP4 or its antagonist, noggin, were grown on agarose beads and implanted into the periotic mesenchyme surrounding the chick otocyst. Although the BMP4-producing cells had no effect on the mature inner ear structure when implanted alone, noggin-producing cells implanted adjacent to the BMP4 cell foci prevented normal semicircular canal development. Beads implanted at the anterior BMP4 focus eliminated the anterior and/or the horizontal canals. Noggin cells implanted at the posterior focus eliminated the posterior canal. Canal loss was prevented by co-implantation of BMP4 cell beads next to noggin beads. An antibody to the chick hair cell antigen (HCA) was used to examine sensory cell distribution, which was abnormal only in the affected tissues of noggin-exposed inner ears. These data suggest a role for BMP4 in the accurate and complete morphological development of the semicircular canals.     

The cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophin in the developing inner ear of the zebrafish, Danio rerio

Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development.     

Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells

Although neural crest cells are known to be very responsive to environmental cues during their development, recent evidence indicates that at least some subpopulations may be committed to a specific differentiation program prior to migration. Because the neural crest is composed of a heterogeneous mixture of cells that contributes to many vertebrate cell lineages, assessing the properties of specific subpopulations and the effect of the environment on their development has been difficult. To address this problem, we have isolated a pure subpopulation of chick mesencephalic neural crest cells by fluorescence no-flow cytometry after labeling them with monoclonal antibodies (Mabs) to a 75-kDa cell surface antigen that is associated with high affinity choline uptake. When cultures of chick mesencephalic neural crest cells are labeled with these Mabs and a fluorescent second step antibody, approximately 5% of the cells are antigen-positive (A+). After sorting, 100% of the resulting cultured mesencephalic neural crest cells are A+. The Mabs we used also label all of the neurons of the embryonic chick and quail ciliary ganglion in vivo and in vitro. We have compared the effect of various cell culture media on the isolated neural crest subpopulation and the heterogeneous chick mesencephalic neural crest from which it was derived. A+ cells were passaged and grown in a variety of media, each of which differently affected its characteristics and development. A+ cells proliferated in the presence of 15% fetal bovine serum (FBS) and high concentrations (10-15%) of chick embryo extract, but did not differentiate, although they retained basal levels of choline acetyltransferase (ChAT) activity. However, in chick serum and high (25 mM as opposed to 7 mM) K+, and heart-, iris-, or lung-conditioned medium, all of which are known to promote survival and/or cholinergic development of ciliary ganglion neurons, the cells ceased to proliferate and all of the cells in the culture became "neuron-like" within 10 days. No neuron-like cells were found in liver-, notocord-, or neural tube-conditioned media if FBS was used. When A+ cells were eliminated either by complement-mediated cytotoxicity or by laser-ablating A+ cells during no-flow cytometry, all ChAT activity was also eliminated, and no neuron-like cells or ChAT activity was found in cultures during a subsequent 3-week culture period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.Download video file.^{(61M, mov)}