Optophysiological analysis of associational circuits in the olfactory cortex

Primary olfactory cortical areas receive direct input from the olfactory bulb, but also have extensive associational connections that have been mainly studied with classical anatomical methods. Here, we shed light on the functional properties of associational connections in the anterior and posterior piriform cortices (aPC and pPC) using optophysiological methods. We found that the aPC receives dense functional connections from the anterior olfactory nucleus (AON), a major hub in olfactory cortical circuits. The local recurrent connectivity within the aPC, long invoked in cortical autoassociative models, is sparse and weak. By contrast, the pPC receives negligible input from the AON, but has dense connections from the aPC as well as more local recurrent connections than the aPC. Finally, there are negligible functional connections from the pPC to aPC. Our study provides a circuit basis for a more sensory role for the aPC in odor processing and an associative role for the pPC

Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2

Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a β strand within the μ–homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 β strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding–defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals

Visualisierung rezyklierender synaptischer Vesikel-Proteine während der Exo-Endozytose an einzelnen hippokampalen Boutons

Während der Exozytose werden synaptische Vesikelproteine in die Plasmamembran freigesetzt und müssen durch kompensatorische Endozytose wieder zurückgewonnen werden. Wenn man GFP-markierte Varianten der Vesikelproteine Synaptobrevin 2 / VAMP 2 und Synaptotagmin 1 in Neuronen überexprimiert, befinden sich davon unter Ruhebedingungen bis zu 30% auf den axonalen Außenmembranen. Um zu testen, ob und zu welchem Außmaß diese in der Plasmamembran gestrandeten Proteine am exo-endozytotischen Zyklieren teilnehmen, wurde ein neuartiger proteolytischer Ansatz angewendet, der es erlaubt deren Schicksal visuell von dem der Proteine zu unterscheiden, die bei der Fusion neu hinzugefügt werden. Die gewonnenen Daten deuten darauf hin, dass sich beide Pools rasch durch Diffusion vermischen und dass endozytierte Vesikel größtenteils aus vorherig gestrandeten Proteinen zusammengesetzt sind. Das Ausmaß an Nicht-Identität exo- und endozytierter vesikulärer Proteine ist von der Stimulationsdauer abhängig. Durch Coexpression von Synaptophysin 1 und durch den Einsatz eines Antikörpers gegen die externe luminale Domäne Synaptotagmins 1 konnte geschätzt werden, dass sich unter physiologischen Bedingungen etwa 10% der vesikulären Proteine nah an der aktiven Zone befinden, wovon diese präferenziell wiederaufgenommen werden, sobald die Stimulation einsetzt. Diese Befunde legen nahe, dass der sofort-freisetzbare-Pool an Vesikeln auf der endozytotischen Seite des Zyklus durch einen sofort-aufnahmebereiten-Pool an teilweise vorassemblierten Vertiefungen ausgeglichen sein könnte, aus dem sich schnell neue Vesikel bilden können, so daß die Aufrechterhaltung des rezyklierenden Vesikel-Pools unter normalen Stimulationsbedingungen gewährleistet ist.Zweitens wurde in Kollaboration mit dem Labor Prof. Dr. Volker Hauckes (Institut für Chemie und Biochemie, Freie-Universität-Berlin) der Zusammenhang zwischen der Funktion des Stonin 2-Proteins in Clathrin-vermittelter Endozytose und Synaptotagmin1-Rückgewinnung untersucht. Obwohl Clathrin-vermittelte Endozytose ein Hauptweg des präsynaptischen Vesikel-Zyklierens zu sein scheint, konnte in Säugetieren bisher kein Sortierungsadapter identifiziert werden, der der Endozytose synaptischer Vesikelmembran-Proteine zugeordnet ist. Gemeinsam konnten wir aufzeigen, dass Stonin 2 ein AP-2-abhängiger Sortierungsadapter für die endozytotische Internalisierung und Rückgewinnung von Synaptotagmin 1 in Neuronen ist

Population imaging at subcellular resolution supports specific and local inhibition by granule cells in the olfactory bulb

Information processing in early sensory regions is modulated by a diverse range of inhibitory interneurons. We sought to elucidate the role of olfactory bulb interneurons called granule cells (GCs) in odor processing by imaging the activity of hundreds of these cells simultaneously in mice. Odor responses in GCs were temporally diverse and spatially disperse, with some degree of non-random, modular organization. The overall sparseness of activation of GCs was highly correlated with the extent of glomerular activation by odor stimuli. Increasing concentrations of single odorants led to proportionately larger population activity, but some individual GCs had non-monotonic relations to concentration due to local inhibitory interactions. Individual dendritic segments could sometimes respond independently to odors, revealing their capacity for compartmentalized signaling in vivo. Collectively, the response properties of GCs point to their role in specific and local processing, rather than global operations such as response normalization proposed for other interneurons

341 Repeats is Not Enough for Methylation in a New Fragile X Mouse Model

AbstractFragile X syndrome (FXS) is a leading monogenic cause of intellectual disability and autism spectrum disorders, spurring decades of intense research and a multitude of mouse models. So far, these models do not recapitulate the genetic underpinning of classical FXS—CGG repeat-induced methylation of theFmr1locus—and their findings have failed to translate into the clinic. We sought to answer whether this disparity was because of low repeat length and generated a novel mouse line with 341 repeats,Fmr1hs341, which is the largest allele in mice reported to date. This repeat length is significantly longer than the 200 repeats generally required for methylation of the repeat tract and promoter region in FXS patients, which leads to silencing of theFMR1gene. Bisulfite sequencing fails to detect the robust methylation expected of FXS inFmr1hs341mice. Quantitative real-time PCR and Western blotting results also do not resemble FXS and instead produce a biochemical profile consistent with the fragile X-associated premutation disorders. These findings suggest that repeat length is unlikely to be the core determinant preventing methylation in mice, and other organisms phylogenetically closer to humans may be required to effectively model FXS.</jats:p

Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion

Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2

Neurotransmission depends on the exocytic fusion of synaptic vesicles (SVs) and their subsequent reformation either by clathrin-mediated endocytosis or budding from bulk endosomes. How synapses are able to rapidly recycle SVs to maintain SV pool size, yet preserve their compositional identity, is poorly understood. We demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) in mice compromises the fidelity of SV protein sorting, whereas the apparent speed of SV retrieval is increased. Loss of Stn2 leads to selective missorting of synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated SV protein endocytosis. The latter phenotype is mimicked by overexpression of endocytosis-defective variants of synaptotagmin 1. Increased speed of SV protein retrieval in the absence of Stn2 correlates with an up-regulation of SV reformation from bulk endosomes. Our results are consistent with a model whereby Stn2 is required to preserve SV protein composition but is dispensable for maintaining the speed of SV recycling