J. Meza is supported by fellowships from Mitacs and Research Manitoba. Funding for this work was provided by Canadian Institutes for Health Research, Research Manitoba, Manitoba Medical Service Foundation, start-up funding from the University of Manitoba and Brain Canada through the Canada Brain Research Fund, with the financial support of Health Canada and the Azrieli Foundation. The views expressed herein do not necessarily represent the views of the Minister of Health or the Government of Canada.

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+pericytes+regulate+cerebral+blood+flow+in+health+and+disease.">Capillary pericytes regulate cerebral blood flow in health and disease.</a> Nature. 508 (1), 55-60 (2014).</li><li>Nilsson, H., Aalkjaer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasomotion:+mechanisms+and+physiological+importance.">Vasomotion: mechanisms and physiological importance.</a> Molecular interventions. 3 (2), 79-89 (2003).</li><li>Rungta, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+arbors+in+layer+II+/+III+somatosensory+cortex.">Vascular arbors in layer II / III somatosensory cortex.</a> Communications Biology. , (2021).</li></ol>

The authors have nothing to disclose.

The present method provides details on mouse tail vein injection with a catheter, two-photon microscope image acquisition for depth stacks, cell calcium signaling movies, creation of hemodynamic kymographs, and calcium and hemodynamic analysis with our image processing algorithms17 (Figure 1). There are several advantages to these techniques that improve the in vivo imaging outcome and reduce time, resources, and animal stress during the session. First, the use of a catheter for tail vein injection provides more control over the needle, the syringe and the amount of substance injected into the circulation of the mouse. Additionally, it prevents dye injection into the tail tissue, saving expensive reagents. Second, we use transgenic mice which express genetically encoded calcium sensors in ensheathing pericytes and demonstrate how to localize them within the brain vascular network with a depth z-stack, which facilitates cell identification and relocation in subsequent imaging sessions long-term. This is an important factor in pericyte studies and ensures proper cell classification6,7. Third, we provide our parameters for collecting calcium movies and hemodynamic line scan data which are a good starting point for measuring dynamic cellular signals. Finally, we present our image processing algorithms17, a comprehensive image processing toolbox which contains multiple approaches for image pre-processing (such as spectral unmixing), calcium image analysis, and hemodynamic analysis (diameter, velocity, etc.). These algorithms can generate plots for a quick and easy visualization of the data, while minimizing the level of user expertise required to analyze results. Furthermore, it can be automated with a few lines of code to quickly batch process multiple datasets with the same parameters. This can potentially improve data visualization and the time investment of the researcher.
The key to collecting good calcium imaging data is to adjust the laser power and PMT settings for clear fluorescence signal acquisition, but to also collect data at a sufficient frame rate to capture the entire calcium event. The data in this protocol was acquired at 10-11 frames per second, which captures the slower calcium oscillations in ensheathing pericytes. There are also several steps during analysis that can improve the analysis outcome. First, spectral unmixing is beneficial if there is significant overlap between the emission spectra of fluorophores (Figure 2). Fluorescein-dextran was used in this protocol because it is a cost-effective and commercially available dextran conjugate that is commonly used for hemodynamic measurements5. Spectral unmixing helps to clean up the data for enhanced detection of calcium signals, but alternative fluorophores with narrower emission spectra could also be used. Second, hand selecting cellular structures as ROIs (Figure 3) is useful for classifying calcium events in different sub-cellular regions such as the soma or process branches. Activity-based ROI selection (Figure 4)16 provides more spatial and temporal information about individual calcium events. This can be helpful when determining the frequency of calcium events in a given area or the propagation of events to other cellular areas. The use of programming software to analyze imaging data can save researchers hours of time when data is batch-processed, but it requires some initial time investment to adjust the parameters for optimal results. The most important factors are the expected size (in &#181;m2) of the active region as well as the duration of the signal (minimum signal time and maximum signal time must be defined). Researchers must examine some example T-series movies first to best determine which parameters fit their data. Finally, poor quality data acquired on the microscope can greatly hinder the analysis of calcium and hemodynamics (Figure 6). Therefore, care should be taken to optimize the microscope acquisition settings in the beginning. With these factors in mind, this protocol that can be adapted to fit calcium imaging or analysis of other dynamic cellular signals (e.g., fluorescent sodium, potassium, metabolite, or voltage fluctuations) in other tissues or cell types.
There are several limitations to this protocol. First, the data is collected under anesthesia, which affects brain activity and could impact blood flow. Similar imaging can be done in awake mice that are trained to accept head fixation for more physiological results. Additionally, it is important to remember that we collect 2-dimensional images of a 3-dimensional cell and blood vessel in vivo. Therefore, we can only capture a faction of the calcium events within these cells or the blood flow in a single section of blood vessel at a time.
Another limitation to note is that two-photon calcium imaging is sensitive to motion artifacts, where movement in and out of the focal plane can be mistaken for calcium fluctuations. This protocol was performed under anesthesia, which limits movement of the animal; however, motion artifacts can be introduced by the breathing rate of the mouse, heart rate, possible tissue swelling, and in the case of ensheathing pericytes, vessel contraction or vasomotion 4,6,18,19. Motion artifacts can be mitigated by several strategies. The image processing packages used in this protocol include an optional motion correction step, which utilizes a 2D convolution engine to align the images within the T-series based on the visible vasculature13,17. Frames with significant changes in the focal plane are identified by this algorithm and can be excluded from analysis. Additionally, it is possible to use statistical strategies within the imaging processing packages, such as a Z-score when generating the fluorescence traces to normalize the movement induced calcium fluctuations20. The most robust approach to account for motion artifacts in two-photon imaging is to combine expression of two fluorescent indicators within the same cell, such as a calcium indicator (e.g., GCaMP) and a fluorescent reporter (e.g., mCherry) that is calcium-independent. Fluctuations in the fluorescent reporter can then be attributed to movement and are subtracted from the calcium indicator signal to normalize motion artifacts.
The purpose of this protocol is to provide a clear understanding of how to collect optimal calcium imaging and blood flow data in vivo and to present new methods and analysis tools that researchers can implement in order to improve their results. These techniques can be applied to study the role of different pericytes populations in blood flow control or in different brain disease states. These imaging parameters can also be used to study calcium and blood flow in other cell types and organ systems and similar principles apply to other dynamic imaging techniques that are made possible by other genetically encoded sensors, beyond calcium.

The central nervous system vasculature consists of penetrating arterioles, capillaries, and ascending venules. Within this network, mural cells such as vascular smooth muscle cells encase arterioles and pericytes extend cellular processes along the first arteriole branches and capillaries1. Pericytes appear to have several roles within the brain including maintenance of the blood-brain-barrier1,2, migration and motility3, potential stem-cell properties, and the regulation of brain blood flow4,5,6. Many of the functional roles of pericytes have been linked to fluctuations in intracellular calcium that may regulate the dilation or contraction of these cells4,5,6.
Several recent studies have set criteria for identifying different types of brain pericytes7,8. Mural cells within the first 4 branches of penetrating arterioles are ensheathing pericytes based on their expression of the contractile protein &#945;-smooth muscle actin (&#945;SMA) and their protruding, ovoid somata with processes that wrap around vessels7,8,9. To visualize calcium fluctuations in ensheathing pericytes, this protocol uses a novel transgenic mouse line, Acta2-RCaMP1.07, also known as Tg(RP23-370F21-RCaMP1.07)B3-3Mik/J10. These mice express the red genetically encoded calcium indicator, RCaMP1.07, in &#945;SMA expressing cells (vascular smooth muscle cells and ensheathing pericytes). Breeding colonies are maintained by crossing noncarrier animals with hemizygotes. RCaMP1.07 is a red fluorescent protein with a calmodulin binding domain, which increases fluorescence when binding to the intracellular calcium10,11. This protocol outlines the steps for combined calcium imaging of ensheathing pericytes and blood flow measurements by two photon microscopy including procedures for tail vein injection of fluorescent dyes, microscope image acquisition in anesthetized mice, and data analysis with programming platforms (Figure 1). These techniques are useful for addressing questions about mural cell physiology but can be adapted to study calcium transients in any cell type in the brain or other organ system.
A 10-month-old female Acta2-RCaMP1.07 mouse was used for the experiment presented in this article. The mouse underwent surgery for chronic cranial window and head post implantation two months prior. Details for the surgical protocol are discussed in previous studies12,13 and similar procedures have been performed in other previously published protocols14,15. The vasculature is labeled with green fluorescein-Dextran (70,000 MW, anionic solution, 2.5% w/v) injected intravenously. This dye is cost effective and readily available from commercial sources, but it has a wider emission spectrum that may overlap with RCaMP emission and bleed through during microscope image&#160;acquisition. Steps for spectral unmixing are outlined in Section 4 below to circumvent this, but other green dyes with narrower emission spectra, such as those based on EGFP, may also be used.

<table><tbody><tr><td>Acta2-RCaMP1.07</td><td>The Jackson Laboratory</td><td>28345</td><td>In the video protocol the animal model used is a female mouse&amp;nbsp; of&amp;nbsp; 10 months, 1 day old.</td></tr><tr><td>Applicators (Regular)</td><td>Bisco</td><td>X-80250P</td><td></td></tr><tr><td>BioFormats package for MATLAB</td><td>NA</td><td>NA</td><td>Denominated in this protocol as &quot;image processing packages&quot;.&amp;nbsp; Available in: https://docs.openmicroscopy.org/bio-formats/</td></tr><tr><td>CHIPS MATLAB toolbox</td><td>NA</td><td>NA</td><td>Denomitaded in this protocol as &quot;image processing algorithms&quot;. Barrett MJP, Ferrari KD, Stobart JL, Holub M, Weber B. CHIPS: an Extensible Toolbox for Cellular and Hemodynamic Two-Photon Image Analysis. Neuroinformatics. 2018;16(1):145-147. doi:10.1007/s12021-017-9344-y. Available in: https://github.com/EIN-lab/CHIPS</td></tr><tr><td>Clear Ultrasound Gel, Medium viscosity</td><td>HealthCare Plus</td><td>UGC250</td><td></td></tr><tr><td>Dextran, fluorescein, 70,000 MW, anionic</td><td>Thermo Fisher Scientific</td><td>D1823</td><td></td></tr><tr><td>Dextran, Texas Red, 70,000 MW, neutral</td><td>Thermo Fisher Scientific</td><td>D1830</td><td></td></tr><tr><td>Eye Lube Plus</td><td>Optixcare</td><td>NA</td><td></td></tr><tr><td>FIJI</td><td>Image J</td><td>NA</td><td>Denominated in this protocol as &quot;image processor software&quot;.&amp;nbsp; Available in: https://imagej.net/Fiji/Downloads</td></tr><tr><td>GCaMP6sfl/fl</td><td>The Jackson Laboratory</td><td></td><td></td></tr><tr><td>Head Post fixing platform</td><td>University of Zurich</td><td>NA</td><td></td></tr><tr><td>Ketamine (Narketan 100 mg/mL)</td><td>Vetoquinol</td><td>440893</td><td></td></tr><tr><td>MATLAB R2020b</td><td></td><td>NA</td><td>Denominated in this protocol as &quot;programming platform &quot;. Available in: https://www.mathworks.com/downloads/</td></tr><tr><td>Needle 0.3mmx25mm</td><td>BD PrecisionGlide</td><td>305128</td><td></td></tr><tr><td>Objective XLUMPLFLN20XW</td><td>Olympus</td><td>NA</td><td>https://www.olympus-lifescience.com/en/objectives/lumplfln-w/</td></tr><tr><td>PDGFR&amp;beta;-CreERT2</td><td>The Jackson Laboratory</td><td>30201</td><td></td></tr><tr><td>Polyethylene Tubing, PE10 I.D. 28mm (0.11&amp;rdquo;) O.D. 61mm (.024&amp;rdquo;)</td><td>BD Intramedic</td><td>427401</td><td></td></tr><tr><td>Prairie View</td><td>Bruker Fluorescence Microscopy</td><td>NA</td><td>https://www.bruker.com/en/products-and-solutions/fluorescence-microscopy/multiphoton-microscopes/ultima-in-vitro.html</td></tr><tr><td>Ultima In Vitro Multiphoton Microscope</td><td>Bruker Fluorescence Microscopy</td><td>NA</td><td>https://www.bruker.com/en/products-and-solutions/fluorescence-microscopy/multiphoton-microscopes/ultima-in-vitro.html</td></tr><tr><td>Under Tank Heater</td><td>Reptitherm U.T.H</td><td>E169064</td><td></td></tr><tr><td>Xylazine (Rompun 20 mg/mL)</td><td>Bayer HealthCare</td><td>2169592</td><td></td></tr></tbody></table>

brain pericyte calcium and hemodynamic imaging in transgenic mice in vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Fluorescein-dextran has a broad emission spectrum that can bleed through into the red channel, impacting RCaMP detection in ensheathing pericytes (Figure 2A). Spectral unmixing after data acquisition in the software program reduces the fluorescein bleeding through (Figure 2B, lower), enhancing calcium signal detection in subsequent analysis steps.
Calcium analysis with the image processing algorithms used in this protocol allows several different approaches to identify ROIs and intracellular calcium fluctuations (i.e., calcium signals). Selecting cellular structures by hand permits detection of calcium fluctuations within these regions (Figure 3A), including different types of signal peaks, such as single peaks and multi-peaks, after the normalized calcium traces are low-pass and band-pass filtered (Figure 3B). Additionally, ROIs are identified by grouping active pixels together where the fluorescence intensity changes over time using image processing algorithms developed by Ellefsen et al. 201416 and Barrett et al. 201817 (Figure 4). This can be applied to any dynamic cellular signal by adjusting the time, threshold and spatial parameters to encompass the expected size and shape of the signal. Decreasing the threshold for signal identification finds more regions of interest (Figure 4B).
Bright and clear hemodynamic kymographs can be analyzed to measure diameter and RBC velocity in blood vessels near ensheathing pericytes (Figure 5A, B). The diameter is calculated from the full width at half maximum of the fluorescence (Figure 5C). The RBC velocity is approximated from the streaks made from unlabeled RBCs, where the angle is input into a Radon transformation to calculate the velocity, flux (cells/s) and linear density (cells/mm; Figure 5D). Poor-quality kymographs where there is fluorescence saturation, poor signal to noise ratio or movement of the imaging field (Figure 6A) creates unreliable plots with error points (red crosses) where data cannot be determined (Figure 6B, C). The quality of the acquired data is critical for a good outcome and following the steps described in this protocol ensures good results.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62725/62725fig01.jpg" /> 
Figure 1. Summary of protocol. The protocol presents the steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. The protocol is divided in 4 steps. 1) Procedure preparation: set up of equipment and catheter preparation; 2) Tail vein injection; 3) Data acquisition by two-photon microscopy; 4) Data analysis with image processing algorithms. <a href="https://www.jove.com/files/ftp_upload/62725/62725fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62725/62725fig02.jpg" /> 
Figure 2. Spectral unmixing of fluorophores. A) Representative average image of RCaMP ensheathing pericyte and fluorescein-dextran labelled blood vessel from a T-series acquisition. Scale bar= 10 &#181;m. B) Upper: When considering individual channels, bleed-through from Channel 2 is apparent in Channel 1 (left). Lower: After spectral unmixing, the bleed-through is reduced and signal from RCaMP is more prominent in the pericyte structure. <a href="https://www.jove.com/files/ftp_upload/62725/62725fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62725/62725fig03.jpg" /> 
Figure 3. Hand selected ROIs and optimized calcium traces. A) Regions of interest selected in the used image processing software (rainbow shapes) can be used to identify calcium signal traces. B) Signal peaks from normalized traces are identified by low pass and band pass filtering the data. We defined the signal threshold as 3 times the standard deviation of the baseline period (first 30 frames) and any peaks over this threshold were considered a signal (lower trace). <a href="https://www.jove.com/files/ftp_upload/62725/62725fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62725/62725fig04.jpg" /> 
Figure 4. Automated, activity-based ROIs for calcium analysis. The same data was analyzed with a threshold of 7 times the standard deviation of the baseline (A) and 3 times the standard deviation of the baseline (B). Decreasing the threshold for identifying active pixels finds more ROIs (B) and signal peaks (pie chart) within the pericytes. <a href="https://www.jove.com/files/ftp_upload/62725/62725fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62725/62725fig05.jpg" /> 
Figure 5. Kymograph hemodynamic measurements. A) Example line scan through the vessel. B) Example of well-defined kymographs for diameter (left) and velocity (right). The black streaks within the right band of fluorescence correspond to RBCs. C) Diameter analysis with clear vasomotion fluctuations. D) Velocity analysis with plots for Y axis= RBC flux(cells/s), line density (cells/mm), velocity (mm/s), and streak angle (degree), signal to noise ratio (arbitrary units, a.u.), X axis=time (sec). <a href="https://www.jove.com/files/ftp_upload/62725/62725fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62725/62725fig06.jpg" /> 
Figure 6. Representation of Poor-Quality Hemodynamic measurements. A) Examples of poor-quality kymographs with fluorescence saturation, poor signal to noise ratio, or movement of the imaging field during acquisition. B and C) Plots similar to Figure 7 of diameter and velocity data that have error points (red dots) because of the poor-quality of the kymographs. (image E, Y axis=Diameter (&#181;m), X axis=time (sec); image F, Y axis= RBC flux(cells/s), line density (cells/mm), velocity (mm/s), and streak angle (degree), signal to noise ratio (a.u.), X axis=time (sec). <a href="https://www.jove.com/files/ftp_upload/62725/62725fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo at JoVE.com

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

brain-pericyte-calcium-hemodynamic-imaging-transgenic-mice

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3.6K Views.  University of Manitoba. De hersenvasculatuur is een complex netwerk van doordringende arteriëlen, haarvaten en oplopende venules. Deze vaten worden omhuld door muurschilderingcellen, zoals ensheathing pericyten, die alfa-gladde spieractine tot expressie brengen, en worden gevonden in de eerste vier takken van penetrerende arteriëlen. We gebruiken Acta2-RCaMP 1.07 transgene muizen om calciumtransiënten in deze cellen te visualiseren, door de expressie van de genetisch gecodeerde calciumindicator RCaMP.Calci...

Video: Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Bioengineering

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Biology

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Medicine

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

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