Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Li, S., Raychaudhuri, S., Lee, S. A., Brockmann, M. M., Wang, J., Kusick, G., Prater, C., Syed, S., Falahati, H., Ramos, R., Bartol, T. M., Hosy, E., & Watanabe, S. Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses. Nature Communications, 12(1), (2021): 677, https://doi.org/10.1038/s41467-021-21004-x.Neurotransmitter is released synchronously and asynchronously following an action potential. Our recent study indicates that the release sites of these two phases are segregated within an active zone, with asynchronous release sites enriched near the center in mouse hippocampal synapses. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release can lead to maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are likely organized to activate NMDA receptors efficiently.e also thank the Marine Biological Laboratory and their Neurobiology course for supporting the initial set of experiments (course supported by National Institutes of Health grant R25NS063307). S.W. and this work were supported by start-up funds from the Johns Hopkins University School of Medicine, Johns Hopkins Discovery funds, and the National Science Foundation (1727260), the National Institutes of Health (1DP2 NS111133-01 and 1R01 NS105810-01A1) awarded to S.W. S.W. is an Alfred P. Sloan fellow, McKnight Foundation Scholar, and Klingenstein and Simons Foundation scholar. G.K. was supported by a grant from the National Institutes of Health to the Biochemistry, Cellular and Molecular Biology Program of the Johns Hopkins University School of Medicine (T32 GM007445) and is a National Science Foundation Graduate Research Fellow (2016217537). E.H. and T.M.B. are supported by CRCNS-NIH-ANR grant AMPAR-T. The EM ICE high-pressure freezer was purchased partly with funds from an equipment grant from the National Institutes of Health (S10RR026445) awarded to Scot C Kuo

Isolation of mouse mammary epithelial subpopulations: a comparison of leading methods

Isolation of mammary epithelial subpopulations, including stem and progenitor cells, has become a standard technique in recent years. However, a number of methods and approaches for this have developed and the relative benefits of the different approaches, and the reason for their development, have not always been clear. Here, three of the leading laboratories working on the separation of mammary cell subpopulations have summarised their methods, highlighted their differences and similarities and also discussed the reasoning behind the approaches they have taken. This article will assist workers establishing mammary cell separation protocols in their laboratories to make informed choices about the methods they should use

Optimization of Process Parameters for a Quasi-Continuous Tablet Coating System Using Design of Experiments

The aim of this study was to identify and optimize the critical process parameters of the newly developed Supercell quasi-continuous coater for optimal tablet coat quality. Design of experiments, aided by multivariate analysis techniques, was used to quantify the effects of various coating process conditions and their interactions on the quality of film-coated tablets. The process parameters varied included batch size, inlet temperature, atomizing pressure, plenum pressure, spray rate and coating level. An initial screening stage was carried out using a 26−1(IV) fractional factorial design. Following these preliminary experiments, optimization study was carried out using the Box–Behnken design. Main response variables measured included drug-loading efficiency, coat thickness variation, and the extent of tablet damage. Apparent optimum conditions were determined by using response surface plots. The process parameters exerted various effects on the different response variables. Hence, trade-offs between individual optima were necessary to obtain the best compromised set of conditions. The adequacy of the optimized process conditions in meeting the combined goals for all responses was indicated by the composite desirability value. By using response surface methodology and optimization, coating conditions which produced coated tablets of high drug-loading efficiency, low incidences of tablet damage and low coat thickness variation were defined. Optimal conditions were found to vary over a large spectrum when different responses were considered. Changes in processing parameters across the design space did not result in drastic changes to coat quality, thereby demonstrating robustness in the Supercell coating process

Isolation of Mouse Mammary Epithelial Subpopulations: A Comparison of Leading Methods

Isolation of mammary epithelial subpopulations, including stem and progenitor cells, has become a standard technique in recent years. However, a number of methods and approaches for this have developed and the relative benefits of the different approaches, and the reason for their development, have not always been clear. Here, three of the leading laboratories working on the separation of mammary cell subpopulations have summarised their methods, highlighted their differences and similarities and also discussed the reasoning behind the approaches they have taken. This article will assist workers establishing mammary cell separation protocols in their laboratories to make informed choices about the methods they should use

Phenotypic and functional characterisation of the luminal cell hierarchy of the mammary gland

Introduction: The organisation of the mammary epithelial hierarchy is poorly understood. Our hypothesis is that the luminal cell compartment is more complex than initially described, and that an understanding of the developmental relationships within this lineage will help in understanding the cellular context in which breast tumours occur. Methods: We used fluorescence-activated cell sorting along with in vitro and in vivo functional assays to examine the growth and differentiation properties of distinct subsets of human and mouse mammary epithelial cells. We also examined how loss of steroid hormones influenced these populations in vivo. Gene expression profiles were also obtained for all the purified cell populations and correlated to those obtained from breast tumours.Results: The luminal cell compartment of the mouse mammary gland can be resolved into nonclonogenic oestrogen receptor-positive (ER+) luminal cells, ER+ luminal progenitors and oestrogen receptor-negative (ER-) luminal progenitors. The ER+ luminal progenitors are unique in regard to cell survival, as they are relatively insensitive to loss of oestrogen and progesterone when compared with the other types of mammary epithelial cells. Analysis of normal human breast tissue reveals a similar hierarchical organisation composed of nonclonogenic luminal cells, and relatively differentiated (EpCAM+CD49f+ALDH-) and undifferentiated (EpCAM+CD49f+ALDH+) luminal progenitors. In addition, approximately one-quarter of human breast samples examined contained an additional population that had a distinct luminal progenitor phenotype, characterised by low expression of ERBB3 and low proliferative potential. Parent-progeny relationship experiments demonstrated that all luminal progenitor populations in both species are highly plastic and, at low frequencies, can generate progeny representing all mammary cell types. The ER- luminal progenitors in the mouse and the ALDH+ luminal progenitors in the human appear to be analogous populations since they both have gene signatures that are associated with alveolar differentiation and resemble those obtained from basal-like breast tumours. Conclusion:The luminal cell compartment in the mammary epithelium is more heterogeneous than initially perceived since progenitors of varying levels of luminal cell differentiation and proliferative capacities can be identified. An understanding of these cells will be essential for understanding the origins and the cellular context of human breast tumours

Early lineage segregation of multipotent embryonic mammary gland progenitors.

The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.SCOPUS: ar.jSCOPUS: er.jinfo:eu-repo/semantics/publishe