scholarly journals Secretory vesicle trafficking in awake and anesthetized mice: differential speeds in axons versus synapses

2018 ◽  
Author(s):  
Johannes Knabbe ◽  
Joris Nassal ◽  
Matthijs Verhage ◽  
Thomas Kuner
Keyword(s):  
Transport Properties ◽  
Mouse Brain ◽  
Dense Core ◽  
Mammalian Brain ◽  
Brain State ◽  
Dense Core Vesicle ◽  
List Type ◽  
Intact Mouse ◽  
Synaptic Localization

AbstractNeuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamo-cortical projections of anesthetized and awake mice. We found an average speed of 1 μm/s, maximal speeds of up to 5 μm/s and a pausing fraction of ~11%. Directionality of transport differed between anesthetized and awake mice. In vivo microtubule +-end extension imaging using Macf18-GFP revealed microtubular growth at 0.12 μm/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (~2.1 μm/s) than retrograde transport (~1.4 μm/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.Key pointsDespite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain.DCVs are transported at similar average speeds in the anesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher.Microtubule +-end extension imaging visualized microtubular growth at 0.12 μm/s and revealed that DCVs were transported faster in the anterograde direction.DCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release.Our work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveal novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality.

scholarly journals Simultaneous high-speed imaging and optogenetic inhibition in the intact mouse brain

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Serena Bovetti ◽  
Claudio Moretti ◽  
Stefano Zucca ◽  
Marco Dal Maschio ◽  
Paolo Bonifazi ◽  
...  
Keyword(s):  
Mouse Brain ◽  
High Speed ◽  
Pyramidal Neurons ◽  
Functional Organization ◽  
Single Photon ◽  
Electrophysiological Recording ◽  
Mammalian Brain ◽  
Cellular Mechanisms ◽  
High Speed Imaging ◽  
Intact Mouse

Abstract Genetically encoded calcium indicators and optogenetic actuators can report and manipulate the activity of specific neuronal populations. However, applying imaging and optogenetics simultaneously has been difficult to establish in the mammalian brain, even though combining the techniques would provide a powerful approach to reveal the functional organization of neural circuits. Here, we developed a technique based on patterned two-photon illumination to allow fast scanless imaging of GCaMP6 signals in the intact mouse brain at the same time as single-photon optogenetic inhibition with Archaerhodopsin. Using combined imaging and electrophysiological recording, we demonstrate that single and short bursts of action potentials in pyramidal neurons can be detected in the scanless modality at acquisition frequencies up to 1 kHz. Moreover, we demonstrate that our system strongly reduces the artifacts in the fluorescence detection that are induced by single-photon optogenetic illumination. Finally, we validated our technique investigating the role of parvalbumin-positive (PV) interneurons in the control of spontaneous cortical dynamics. Monitoring the activity of cellular populations on a precise spatiotemporal scale while manipulating neuronal activity with optogenetics provides a powerful tool to causally elucidate the cellular mechanisms underlying circuit function in the intact mammalian brain.

scholarly journals In vivo Three Photon Imaging of Neuronal Activities from Hippocampus in Intact Mouse Brain

2015 ◽  
Vol 21 (S3) ◽  
pp. 1721-1722
Author(s):  
Tianyu Wang ◽  
Dimitre Ouzounov ◽  
Nicholas G. Horton ◽  
Jean C. Cruz Hernandez ◽  
Danielle Feng ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Intact Mouse ◽  
Photon Imaging

scholarly journals In vivo three-photon microscopy of subcortical structures within an intact mouse brain

2013 ◽  
Vol 7 (3) ◽  
pp. 205-209 ◽  
Author(s):  
Nicholas G. Horton ◽  
Ke Wang ◽  
Demirhan Kobat ◽  
Catharine G. Clark ◽  
Frank W. Wise ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Subcortical Structures ◽  
Intact Mouse

In vivo Three-photon Imaging of Brain Activity from Cortical and Subcortical Neurons in Intact Mouse Brain

2015 ◽  
Author(s):  
Dimitre G. Ouzounov ◽  
Tianyu Wang ◽  
Nicholas G. Horton ◽  
Jean C. Cruz Hernández ◽  
Danielle Feng ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Brain Activity ◽  
Intact Mouse ◽  
Photon Imaging

scholarly journals In Vivo Three-Photon Microscopy of Subcortical Structures within an Intact Mouse Brain

2012 ◽  
Author(s):  
Nicholas G. Horton ◽  
Ke Wang ◽  
Demirhan Kobat ◽  
Frank W. Wise ◽  
Chris Xu
Keyword(s):  
Mouse Brain ◽  
Subcortical Structures ◽  
Intact Mouse

scholarly journals Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

Proteomes ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 42 ◽  
Author(s):  
Megan Miller ◽  
Rashaun Wilson ◽  
TuKiet Lam ◽  
Angus Nairn ◽  
Marina Picciotto
Keyword(s):  
Protein Kinase ◽  
Mouse Brain ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Mammalian Brain ◽  
Phosphorylation Sites ◽  
Nicotinic Acetylcholine ◽  
Hek Cells

Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function.

scholarly journals Cell types and clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics

2021 ◽  
Author(s):  
Michael Ratz ◽  
Leonie von Berlin ◽  
Ludvig Larsson ◽  
Marcel Martin ◽  
Jakub Orzechowski Westholm ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Mouse Brain ◽  
High Throughput ◽  
Thin Sheet ◽  
Cell Types ◽  
Mammalian Brain ◽  
Fate Mapping ◽  
Cell Phenotypes

SummaryThe mammalian brain contains a large number of specialized cells that develop from a thin sheet of neuroepithelial progenitor cells1,2. Recently, high throughput single-cell technologies have been used to define the molecular diversity of hundreds of cell types in the nervous system3,4. However, the lineage relationships between mature brain cells and progenitor cells are not well understood, because transcriptomic studies do not allow insights into clonal relationships and classical fate-mapping techniques are not scalable5,6. Here we show in vivo barcoding of early progenitor cells that enables simultaneous profiling of cell phenotypes and clonal relations in the mouse brain using single-cell and spatial transcriptomics. We reconstructed thousands of clones to uncover the existence of fate-restricted progenitor cells in the mouse hippocampal neuroepithelium and show that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny. By coupling spatial transcriptomics with clonal barcoding, we disentangle migration patterns of clonally related cells in densely labelled tissue sections. Compared to classical fate mapping, our approach enables high-throughput dense reconstruction of cell phenotypes and clonal relations at the single-cell and tissue level in individual animals and provides an integrated approach for understanding tissue architecture.

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