Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called “Direct Detection of Biotin-containing Tags” (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h.

Retinotopic maps are plastic in response to changes in sensory input; however, the experience-dependent instructive cues that organize retinotopy are unclear. In animals with forward-directed locomotion, the predominant anterior to posterior optic flow activates retinal ganglion cells in a stereotyped temporal to nasal sequence. Here we imaged retinotectal axon arbor location and structural plasticity to assess map refinement in vivo while exposing Xenopus tadpoles to visual stimuli. We show that the temporal sequence of retinal activity driven by natural optic flow organizes retinotopy by regulating axon arbor branch dynamics, whereas the opposite sequence of retinal activity prevents map refinement. Our study demonstrates that a spatial to temporal to spatial transformation of visual information controls experience-dependent topographic map plasticity. This organizational principle is likely to apply to other sensory modalities and projections in the brain.

The rapid development of microscopic imaging techniques has greatly facilitated time-lapse imaging of neuronal morphology. However, analysis of structural dynamics in the vast amount of 4-Dimensional data generated by in vivo or ex vivo time-lapse imaging still relies heavily on manual comparison, which is not only laborious, but also introduces errors and discrepancies between individual researchers and greatly limits the research pace. Here we present a supervised 4D Structural Plasticity Analysis (4D SPA) computer method to align and match 3-Dimensional neuronal structures across different time points on a semi-automated basis. We demonstrate 2 applications of the method to analyze time-lapse data showing gross morphological changes in dendritic arbor morphology and to identify the distribution and types of branch dynamics seen in a series of time-lapse images. Analysis of the dynamic changes of neuronal structure can be done much faster and with greatly improved consistency and reliability with the 4D SPA supervised computer program. Users can format the neuronal reconstruction data to be used for this analysis. We provide file converters for Neurolucida and Imaris users. The program and user manual are publically accessible and operate through a graphical user interface on Windows and Mac OSX.

Visual system development requires experience-dependent mechanisms that regulate neuronal structure and function, including dendritic arbor growth, synapse formation, and stabilization. Although RNA binding proteins have been shown to affect some forms of synaptic plasticity in adult animals, their role in the development of neuronal structure and functional circuitry is not clear. Using two-photon time-lapse in vivo imaging and electrophysiology combined with morpholino-mediated knockdown and expression of functional deletion mutants, we demonstrate that the mRNA binding protein, cytoplasmic polyadenylation element binding protein1 (CPEB1), affects experience-dependent neuronal development and circuit formation in the visual system of Xenopus laevis. These data indicate that sensory experience controls circuit development by regulating translational activity of mRNAs.

•Exosomes are a mode of intercellular communication within the nervous system.•Although more widely studied in the context of neurological disease, exosomes may serve a beneficial function during brain circuit development.•Exosome signaling may be dynamically regulated by modulating exosome cargo loading and release from donor cells and by modulating receptivity and signaling in recipient cells.•Elucidation of exosome function in brain development and disease requires the generation of tools to identify and manipulate exosome signaling in the intact brain.Exosomes are small extracellular vesicles that mediate intercellular signaling in the brain without requiring direct contact between cells. Although exosomes have been shown to play a role in neurological diseases and in response to nerve trauma, a role for exosome-mediated signaling in brain development and function has not yet been demonstrated. Here we review data building a case for exosome function in the brain.

Regulation of progenitor cell fate determines the numbers of neurons in the developing brain. While proliferation of neural progenitors predominates during early central nervous system (CNS) development, progenitor cell fate shifts toward differentiation as CNS circuits develop, suggesting that signals from developing circuits may regulate proliferation and differentiation. We tested whether activity regulates neurogenesis in vivo in the developing visual system of Xenopus tadpoles. Both cell proliferation and the number of musashi1-immunoreactive progenitors in the optic tectum decrease as visual system connections become stronger. Visual deprivation for 2 days increased proliferation of musashi1-immunoreactive radial glial progenitors, while visual experience increased neuronal differentiation. Morpholino-mediated knockdown and overexpression of musashi1 indicate that musashi1 is necessary and sufficient for neural progenitor proliferation in the CNS. These data demonstrate a mechanism by which increased brain activity in developing circuits decreases cell proliferation and increases neuronal differentiation through the downregulation of musashi1 in response to circuit activity.Highlights► Neuronal progenitor cell proliferation decreases as neuronal circuits mature ► Visual activity reduces proliferation and promotes differentiation in optic tectum ► Musashi1 expression levels are regulated by activity in developing brain circuits ► Musashi1 is necessary and sufficient for neural cell proliferation in Xenopus CNS

Dendrites, axons, and synapses are dynamic during circuit development; however, changes in microcircuit connections as branches stabilize have not been directly demonstrated. By combining in vivo time-lapse imaging of Xenopus tectal neurons with electron microscope reconstructions of imaged neurons, we report the distribution and ultrastructure of synapses on individual vertebrate neurons and relate these synaptic properties to dynamics in dendritic and axonal arbor structure over hours or days of imaging. Dynamic dendrites have a high density of immature synapses, whereas stable dendrites have sparser, mature synapses. Axons initiate contacts from multisynapse boutons on stable branches. Connections are refined by decreasing convergence from multiple inputs to postsynaptic dendrites and by decreasing divergence from multisynapse boutons to postsynaptic sites. Visual deprivation or NMDAR antagonists decreased synapse maturation and elimination, suggesting that coactive input activity promotes microcircuit development by concurrently regulating synapse elimination and maturation of remaining contacts.Highlights► Rapid synaptic reorganization during circuit assembly ► Concurrent synapse elimination and maturation during microcircuit formation ► Combining in vivo time-lapse imaging and retrospective ultrastructure studies ► Both synapse elimination and maturation are activity dependent