Generating inner ear organoids containing putative cochlear hair cells from human pluripotent stem cells

In view of the prevalence of sensorineural hearing defects in an ageing population, the development of protocols to generate cochlear hair cells and their associated sensory neurons as tools to further our understanding of inner ear development are highly desirable. We report herein a robust protocol for the generation of both vestibular and cochlear hair cells from human pluripotent stem cells which represents an advance over currently available methods that have been reported to generate vestibular hair cells only. Generating otic organoids from human pluripotent stem cells using a three-dimensional culture system, we show formation of both types of sensory hair cells bearing stereociliary bundles with active mechano-sensory ion channels. These cells share many morphological characteristics with their in vivo counterparts during embryonic development of the cochlear and vestibular organs and moreover demonstrate electrophysiological activity detected through single-cell patch clamping. Collectively these data represent an advance in our ability to generate cells of an otic lineage and will be useful for building models of the sensory regions of the cochlea and vestibule

Glial Processes at the Drosophila Larval Neuromuscular Junction Match Synaptic Growth

Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ

Multidimensional Characterization and Differentiation of Neurons in the Anteroventral Cochlear Nucleus

Multiple parallel auditory pathways ascend from the cochlear nucleus. It is generally accepted that the origin of these pathways are distinct groups of neurons differing in their anatomical and physiological properties. In extracellular in vivo recordings these neurons are typically classified on the basis of their peri-stimulus time histogram. In the present study we reconsider the question of classification of neurons in the anteroventral cochlear nucleus (AVCN) by taking a wider range of response properties into account. The study aims at a better understanding of the AVCN's functional organization and its significance as the source of different ascending auditory pathways. The analyses were based on 223 neurons recorded in the AVCN of the Mongolian gerbil. The range of analysed parameters encompassed spontaneous activity, frequency coding, sound level coding, as well as temporal coding. In order to categorize the unit sample without any presumptions as to the relevance of certain response parameters, hierarchical cluster analysis and additional principal component analysis were employed which both allow a classification on the basis of a multitude of parameters simultaneously. Even with the presently considered wider range of parameters, high number of neurons and more advanced analytical methods, no clear boundaries emerged which would separate the neurons based on their physiology. At the current resolution of the analysis, we therefore conclude that the AVCN units more likely constitute a multi-dimensional continuum with different physiological characteristics manifested at different poles. However, more complex stimuli could be useful to uncover physiological differences in future studies

Role of calcium alginate and mannitol in protecting Bifidobacterium

Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (aw) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl2 solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The aw of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without aw adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low aws. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low aws. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C)