Disappearance of afferent and efferent nerve terminals in the inner ear of the chick embryo after chronic treatment with beta-bungarotoxin

Beta-Bungarotoxin(beta-BT) was applied to chick embryos at 3-day intervals beginning on the 4th day of incubation to see the effect of chronically and massively applied beta-BT, and to investigate the hair cell-nerve relationship in the developing inner ear by electron microscopy. On the 10th day of incubation, nerve terminals had achieved contact with differentiating hair cells, but the acoustico-vestibular ganglion cells of treated animals were decreased in number to one-third of those of the control. By the 14th day, most of the ganglion cells degenerated and disappeared, and only a few nerve terminals were seen in the neuroepithelium. At this time, most of the hair cells lacked synaptic contacts with nerve terminals; but their presynaptic specialization remained intact and they showed evidence of continuing differentiation. On the 17th day, the acoustico-vestibular ganglion cells were completely absent. All the hair cells were devoid of afferent and efferent innervation but were fully differentiated on the 21st day. Beta-BT was found to have a similar destructive effect on cultured spinal ganglion cells. The present study shows that beta-BT kills acoustico-vestibular and spinal nerve cells when applied chronically and massively during development. Furthermore, the differentiation of hair cells proceeds normally, and their presynaptic specializations are maintained when nerve terminals are absent during later developmental stages.     

Structural variation in the inner ears of four deep-sea elopomorph fishes

Deep-sea fishes have evolved in dark or dimly lit environments devoid of the visual cues available to shallow-water species. Because of the limited opportunity for visual scene analysis by deep-sea fishes, it is reasonable to hypothesize that the inner ears of at least some such species may have evolved structural adaptations to enhance hearing capabilities in lieu of vision. As an initial test of this hypothesis, scanning electron microscopy was used to examine the structure of the inner ears of four deep-sea elopomorph species inhabiting different depths: Synaphobranchus kaupii, Synaphobranchus bathybius, Polyacanthonotus challengeri, and Halosauropsis macrochir. The shape of the sensory epithelia and hair cell ciliary bundle orientation of the saccule, lagena, and utricle, the three otolithic organs associated with audition and vestibular function, are described. The saccules of all four species have a common, alternating ciliary bundle orientation pattern. In contrast, the lagena exhibits more interspecific diversity in shape and ciliary bundle orientation, suggesting that it has special adaptations in these species. The macula neglecta, a sensory epithelium of unknown function, is present in all four species.     

Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells

Chick limb-bud mesoderm cells from embryonic stages 22–25 were cultured at high cell densities in media known to support chondrogenesis. Under these conditions the continuous presence of normal mouse serum, at a concentration of 10%, inhibits the ability of the cells to produce toluidine blue-stainable cartilage matrix materials. In contrast, mesodermal cells treated with comparable concentrations of other heterologous sera continue to differentiate much like the control cultures while growing in the presence of the test sera. The inhibitory effect of the serum was shown not to be the result of a general cytotoxic effect on protein synthesis or the inability of the cells to incorporate [³H]d-glucosamine. There was a significant difference however, in the distribution of the incorporated glucosamine. Less label was associated with the cell layer of the treated series, while a greater amount of the incorporated material was found to be secreted into the medium when compared with the control cultures. Studies have shown also that the serum inhibitory response is dose dependent, while the factor(s) itself is non-dialysable, stable to heat and repeated freezing and is not a conventional serum lipoprotein. Following the addition of whole or delipidated mouse serum, a significant increase in lipid droplets appears in the cytoplasm of the cells. Biochemical analyses of mouse serum-treated cells indicate that there is a marked increase in their triglyceride content as compared to the control cells. While the nature of the serum inhibitory factor remains to be determined, the accumulation of triglyceride following mouse serum treatment suggests that this may play a role in modulating the expression of the chondrogenic phenotype.     

Presynaptic tubular structures in photoreceptor cells

Summary After the application of fixatives including phosphotungstic acid or a mixture of osmium tetroxide and zinc iodide, complex tubular structures are evident in the presynaptic side of the synapses between photoreceptor and bipolar cells of the rat's retina. In the first case only the limiting membranes are visualized, while in the second only the content of the tubules is stained. These tubules seem to be related, on a morphological ground, with the formation of synaptic vesicles. These tubular structures are not observed when fixation is done with osmium tetroxide or glutaraldehyde-osmium tetroxide.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and from National Institutes of Health, U.S.A., (5 RO1 NS 06953-05 NEUA).We want to express our gratitude to Mrs. Haydée Agoff de Zimman and Mr. Alberto Saénz for their skillful technical assistance.     

Presynaptic FMRP and local protein synthesis support structural and functional plasticity of glutamatergic axon terminals

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Evolution of vertebrate mechanosensory hair cells and inner ears: toward identifying stimuli that select mutation driven altered morphologies

Among the major distance senses of vertebrates, the ear is unique in its complex morphological changes during evolution. Conceivably, these changes enable the ear to adapt toward sensing various physically well-characterized stimuli. This review develops a scenario that integrates sensory cell with organ evolution. We propose that molecular and cellular evolution of the vertebrate hair cells occurred prior to the formation of the vertebrate ear. We previously proposed that the genes driving hair cell differentiation were aggregated in the otic region through developmental re-patterning that generated a unique vertebrate embryonic structure, the otic placode. In agreement with the presence of graviceptive receptors in many vertebrate outgroups, it is likely that the vertebrate ear originally functioned as a simple gravity-sensing organ. Based on the rare occurrence of angular acceleration receptors in vertebrate outgroups, we further propose that the canal system evolved with a more sophisticated ear morphogenesis. This evolving morphogenesis obviously turned the initial otocyst into a complex set of canals and recesses, harboring multiple sensory epithelia each adapted to the acquisition of a specific aspect of a given physical stimulus. As support for this evolutionary progression, we provide several details of the molecular basis of ear development.     

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D_Davis (Hb D_D), an autosomal codominant in chickens, the α^D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D_D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M_D), on high-resolution isoelectric focusing gels. Comparison of $\text{P1}\text{P2}$ ratios in the four phenotypes indicated that homozygous Hb D_D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D_D) than did red cells from corresponding adult birds. Heterozygous Hb D_D embroys and adults had both Hb D and Hb D_D. Hb D_D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D_D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D_D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D_D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.     

Building inner ears: recent advances and future challenges for in vitro organoid systems

While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into “inner ear organoids” containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications.Subject terms: Cell biology, Somatic system, Stem-cell research     

Evidence for the presence of precursor B cells in normal and in hormonally bursectomized chick embryos

Embryonic bone marrow of normal and hormonally bursectomized chicks was examined for the presence of hematopoietic precursor cells capable of migrating to the thymus and bursa and of differentiating into functional T and B cells, respectively. Following transfer of chromosomally marked bone marrow of normal and in ovo bursectomized 14-day-old embryos to 14-day-old γ-irradiated embryonic recipients, donor cells proliferated in the marrow, thymus, and bursa of recipients, and differentiated to PHA- and Con A-responsive T cells as well as to dextran sulfate- and anti-immunoglobulin-responsive B cells. In contrast, when marrow of 2-day-old hatched normal and in ovo-bursectomized donors was transferred to 14-day-old embryonic recipients, donor cells repopulated only the marrow and thymus of recipients which was followed by differentiation to Con A- or PHA-responsive T cells, but the same donor cells failed to proliferate in the bursa and there was no differentiation to functional B cells of donor type. The data were fitted to a model of T- and B-cell differentiation from the stem cell level and they suggest the presence of separate populations of committed precursor T (PT) and precursor B (PB) cells in the marrow of normal and in ovo bursectomized embryos with a bursa-independent selective disappearance of PB cells from the marrow during the late embryonic period.     

Presynaptic mechanisms underlying the alpha-lipoic acid facilitation of glutamate exocytosis in rat cerebral cortex nerve terminals

The antioxidant alpha-lipoic acid has been reported to prevent and reverse age-related impairments in learning and memory. However, it is unclear how alpha-lipoic acid improves cognitive function. In this study, the effect of alpha-lipoic acid on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes) was examined. We found that alpha-lipoic acid potently facilitated 4-aminopyridine (4AP)-evoked glutamate release, and this release facilitation results from an enhancement of vesicular exocytosis and not from an increase of non-vesicular release. Examination of the effect of alpha-lipoic acid on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release was associated with an increase in voltage-dependent Ca(2+) influx. Consistent with this, alpha-lipoic acid-mediated facilitation of glutamate release was completely prevented in synaptosomes pretreated with a wide spectrum blocker of the N- and P/Q-type Ca(2+) channels, omega-conotoxin MVIIC. The facilitatory effect of alpha-lipoic acid on Ca(2+) influx was not due to an increase of synaptosomal excitability because alpha-lipoic acid did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. In addition, both ionomycin and hypertonic sucrose-induced glutamate release were enhanced by alpha-lipoic acid. Furthermore, disruption of cytoskeleton organization with cytochalasin D occluded the facilitatory effect of alpha-lipoic acid on 4AP or ionomycin-evoked glutamate release. These results suggest that the antioxidant alpha-lipoic acid enhances the Ca(2+) entry through presynaptic N- and P/Q-type Ca(2+) channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes. Also, activation of PKA and PKC may underlie, at least in part, the alpha-lipoic acid-mediated facilitation of glutamate release observed here as alpha-lipoic acid-enhanced 4AP and ionomycin-evoked glutamate release were significantly attenuated by PKA and PKC inhibitors. This finding may provide some information regarding the mechanism of action of alpha-lipoic acid in the central nervous system (CNS).     

Collagen proteins of human hyaline cartilage in normal states and in various bone dysplasias

Using SDS electrophoresis and subsequent densitometry, isolated collagen proteins of infantile rib and knee joint hyaline cartilage were characterized. Both the normal samples and hyaline cartilages of children with osteochondrodysplasias were shown to contain collagens type I and II as well as collagen proteins with Mr 160 (A), 150 (B), 140 (C), 120 (D), 110 (E) and 39 kD (F), whose content in normal samples varied, depending on the donor age. An analysis of normal and pathological samples revealed the following biochemical markers of intensive chondrocyte proliferation: an increased content of collagen proteins A--F and a decreased number of intramolecular cross-links of collagen type II. Conversely, the increased number of intramolecular cross-links in collagen type II and the elevation of the relative content of collagen type I in lethal forms of osteochondrodysplasias and funnel chest may testify to chondrocyte dedifferentiation. It was assumed that collagen proteins D and E correspond to proteins 1 alpha and 2 alpha, whereas proteins A, B, C and F are the products of hydrolysis by pepsin type M of collagen detected previously only in animal cartilages. Mapping of collagen type II CNBr-peptides and electron microscopic analysis of its SLS-form were carried out. The experimental results are suggestive of the involvement of collagen proteins in the pathogenesis of human osteochondridysplasias as well as of the pronounced biochemical heterogeneity of the disease.     

Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.