Multiplex assessment of the positions of odorant receptor-specific glomeruli in the mouse olfactory bulb by serial two-photon tomography

In the mouse, axons of olfactory sensory neurons (OSNs) that express the same odorant receptor (OR) gene coalesce into one or a few glomeruli in the olfactory bulb. The positions of OR-specific glomeruli are traditionally described as stereotyped. Here, we have assessed quantitatively the positions of OR-specific glomeruli using serial two-photon tomography, an automated method for whole-organ fluorescence imaging that integrates two-photon microscopy with serial microtome sectioning. Our strategy is multiplexed. By repeated crossing, we generated two strains of mice with gene-targeted mutations at four or five OR loci for a total of six ORs: MOR23 (Olfr16), mOR37A (Olfr155), M72 (Olfr160), P2 (Olfr17), MOR256-17 (Olfr15), and MOR28 (Olfr1507). Glomerular imaging relied on intrinsic fluorescence of GFP or DsRed, or on whole-mount immunofluorescence with antibodies against GFP, DsRed, or β-gal using the method of immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). The high-resolution 3D-reconstructed datasets were segmented to identify the labeled glomeruli and to assess glomerular positional variability between the bulbs of one mouse (intraindividual) and among the bulbs of different mice (interindividual). In 26 mice aged 21 or 50 d or 10 wk, we made measurements of the positions of 352 glomeruli. We find that positional variability of glomeruli correlates with the OR: For instance, the medial MOR28 glomerular domain occupies a surface area that is an order of magnitude larger than the surface area of the medial MOR23 glomerular domain. Our results quantify the level of precision that is delivered by the mechanisms of OSN axon wiring, differentially for the various OSN populations expressing distinct OR genes.

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Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2013.201 ◽

2013 ◽

Vol 34 (2) ◽

pp. 325-331 ◽

Cited By ~ 19

Author(s):

Kazuto Masamoto ◽

Hiroyuki Takuwa ◽

Chie Seki ◽

Junko Taniguchi ◽

Yoshiaki Itoh ◽

...

Keyword(s):

Adult Mouse ◽

Fluorescent Protein ◽

Vascular Endothelial Cells ◽

Three Dimensional ◽

Vascular Endothelial ◽

Two Photon Microscopy ◽

Two Photon ◽

Mouse Cortex ◽

Hypoxia Adaptation ◽

Green Fluorescent

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14 μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60 μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

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Three-dimensional imaging of epidermal keratinocytes and dermal vasculatures using two-photon microscopy

Journal of Dermatological Science ◽

10.1016/j.jdermsci.2013.01.005 ◽

2013 ◽

Vol 70 (2) ◽

pp. 143-145 ◽

Cited By ~ 8

Author(s):

Gyohei Egawa ◽

Yohei Natsuaki ◽

Yoshiki Miyachi ◽

Kenji Kabashima

Keyword(s):

Three Dimensional ◽

Epidermal Keratinocytes ◽

Three Dimensional Imaging ◽

Two Photon Microscopy ◽

Two Photon

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Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.15-18491 ◽

2016 ◽

Vol 57 (7) ◽

pp. 3159 ◽

Cited By ~ 3

Author(s):

Xianzeng Zhang ◽

Nenrong Liu ◽

Peng Un Mak ◽

Sio Hang Pun ◽

Mang I. Vai ◽

...

Keyword(s):

Quantitative Measurement ◽

Three Dimensional ◽

Intact Mouse ◽

Two Photon Microscopy ◽

Two Photon ◽

Microscopy Techniques ◽

Three Dimensional Segmentation ◽

Aqueous Outflow ◽

Outflow System

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Bioinformatics Discovery of Putative Enhancers within Mouse Odorant Receptor Gene Clusters

Chemical Senses ◽

10.1093/chemse/bjz043 ◽

2019 ◽

Vol 44 (9) ◽

pp. 705-720

Author(s):

James E Farber ◽

Robert P Lane

Keyword(s):

Odorant Receptor ◽

Receptor Gene ◽

Gene Clusters ◽

Small Subset ◽

Histone 3 ◽

Large Size ◽

Dnase Hypersensitivity ◽

Or Gene ◽

Or Genes ◽

Flanking Regions

Abstract Olfactory neuronal function depends on the expression and proper regulation of odorant receptor (OR) genes. Previous studies have identified 54 putative intergenic enhancers within or flanking 40 mouse OR clusters. At least 2 of these putative enhancers have been shown to regulate the expression of a small subset of proximal OR genes. In recognition of the large size of the mouse OR gene family (~1400 OR genes distributed across multiple chromosomal loci), it is likely that there remain many additional not-as-yet discovered OR enhancers. We utilized 23 of the previously identified enhancers as a training set (TS) and designed an algorithm that combines a broad range of epigenetic criteria (histone-3-lysine-4 monomethylation, histone-3-lysine-79 trimethylation, histone-3-lysine-27 acetylation, and DNase hypersensitivity) and genetic criteria (cross-species sequence conservation and transcription-factor binding site enrichment) to more broadly search OR gene clusters for additional candidates. We identified 181 new candidate enhancers located at 58 (of 68) mouse OR loci, including 25 new candidates identified by stringent search criteria whose signal strengths are not significantly different from the 23 previously characterized OR enhancers used as the TS. Additionally, we compared OR enhancer versus generic enhancer features in order to evaluate likelihoods that new enhancer candidates specifically function in OR regulation. We found that features distinguishing OR-specific function are significantly more evident for enhancer candidates located within OR clusters as compared with those in flanking regions.

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Two-Photon Microscopy of Single Molecules

Microscopy and Microanalysis ◽

10.1017/s1431927600036503 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 802-803

Author(s):

J. T. Fourkas ◽

M. J. R. Previte ◽

R. A. Farrer ◽

C. Olson ◽

L. A. Peyser

Keyword(s):

Near Field ◽

Single Molecules ◽

Three Dimensional ◽

Excitation Beam ◽

Two Photon Microscopy ◽

Two Photon ◽

Higher Power ◽

Excitation Techniques ◽

Field Excitation ◽

Heterogeneous Ensemble

The ability to observe the fluorescence arising from single molecules has revolutionized our ability to study the structure and dynamics of materials on a microscopic level and to probe the properties of individual members of a heterogeneous ensemble. A variety of near-field and far-field excitation techniques have been employed to study single molecules. Multiphoton excitation (MPE) techniques have a number of advantages that make them particularly attractive for singlemolecule detection. First, because the excitation and fluorescence wavelengths are significantly different from one another, Rayleigh and Raman scattering can easily be filtered out, leading to a low number of background counts. Second, because the probability for MPE depends on the excitation intensity to the second or higher power, the excitation is localized to the point in space where the excitation beam is most tightly focussed, thus providing three-dimensional resolution.

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