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The axon initial segment (AIS) is a specialized neuronal compartment involved in the maintenance of axo-dendritic polarity and in the generation of action potentials. It is also a site of significant structural plasticity-manipulations of neuronal activity in vitro and in vivo can produce changes in AIS position and/or size that are associated with alterations in intrinsic excitability. However, to date all activity-dependent AIS changes have been observed in experiments carried out on fixed samples, offering only a snapshot, population-wide view of this form of plasticity. To extend these findings by following morphological changes at the AIS of individual neurons requires reliable means of labeling the structure in live preparations. Here, we assessed five different immunofluorescence-based and genetically-encoded tools for live-labeling the AIS of dentate granule cells (DGCs) in dissociated hippocampal cultures. We found that an antibody targeting the extracellular domain of neurofascin provided accurate live label of AIS structure at baseline, but could not follow rapid activity-dependent changes in AIS length. Three different fusion constructs of GFP with full-length AIS proteins also proved unsuitable: while neurofascin-186-GFP and NaVĪ²4-GFP did not localize to the AIS in our experimental conditions, overexpressing 270kDa-AnkyrinG-GFP produced abnormally elongated AISs in mature neurons. In contrast, a genetically-encoded construct consisting of a voltage-gated sodium channel intracellular domain fused to yellow fluorescent protein (YFP-NaVII-III) fulfilled all of our criteria for successful live AIS label: this construct specifically localized to the AIS, accurately revealed plastic changes at the structure within hours, and, crucially, did not alter normal cell firing properties. We therefore recommend this probe for future studies of live AIS plasticity in vitro and in vivo.
Pubmed ID: 27932952 RIS Download
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View all literature mentionsA national mouse monoclonal antibody generating resource for biochemical and immunohistochemical applications in mammalian brain. NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mouse mAbs that are optimized for use in brain (i.e. NeuroMabs): for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks. The NeuroMab facility was initially funded with a five-year U24 cooperative grant from NINDS and NIMH. The initial goal of the facility for this funding period is to generate a library of novel NeuroMabs against neuronal proteins, initially focusing on membrane proteins (receptors/channels/transporters), synaptic proteins, other neuronal signaling molecules, and proteins with established links to disease states. The scope of the facility was expanded with supplements from the NIH Blueprint for Neuroscience Research to include neurodevelopmental targets, the NIH Roadmap for Medical Research to include epigenetics targets, and NIH Office of Rare Diseases Research to include rare disease targets. These NeuroMabs will then be produced on a large scale and made available to the neuroscience research community on an inexpensive basis as tissue culture supernatants or purified immunoglobulin by Antibodies Inc. The UC Davis/NIH NeuroMab Facility makes NeuroMabs available directly to end users and is unable to accommodate sales to distributors for third party distribution. Note, NeuroMab antibodies are now offered through antibodiesinc.
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