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.

<ol>
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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=Contractile+pericytes+determine+the+direction+of+blood+flow+at+capillary+junctions.">Contractile pericytes determine the direction of blood flow at capillary junctions.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (43), 27022-27033 (2020).</li><li>Hartmann, D. a., 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=Pericyte+structure+and+distribution+in+the+cerebral+cortex+revealed+by+high-resolution+imaging+of+transgenic+mice.">Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice.</a> Neurophotonics. 2 (4), 041402 (2015).</li><li>Grant, R. I., 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=Organizational+hierarchy+and+structural+diversity+of+microvascular+pericytes+in+adult+mouse+cortex.">Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 39 (3), 411-425 (2017).</li><li>Hill, R. A., Tong, L., Yuan, P., Murikinati, S., Gupta, S., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+blood+flow+in+the+normal+and+ischemic+brain+is+controlled+by+arteriolar+smooth+muscle+cell+contractility+and+not+by+capillary+pericytes.">Regional blood flow in the normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not by capillary pericytes.</a> Neuron. 87 (1), 95-110 (2015).</li><li>28345 - STOCK Tg(RP23-370F21-RCaMP1.07)B3-3Mik/J. Jackson Laboratory Available from: <a target="_blank" href="https://www.jax.org/strain/028345">https://www.jax.org/strain/028345</a> (2021)</li><li>Ohkura, M., Sasaki, T., Kobayashi, C., Ikegaya, Y., Nakai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+genetically+encoded+red+fluorescent+Ca2++indicator+for+detecting+optically+evoked+action+potentials.">An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials.</a> PLoS ONE. 7 (7), (2012).</li><li>Stobart, J. 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=Long-term+in+vivo+calcium+imaging+of+astrocytes+reveals+distinct+cellular+compartment+responses+to+sensory+stimulation.">Long-term in vivo calcium imaging of astrocytes reveals distinct cellular compartment responses to sensory stimulation.</a> Cerebral Cortex. 28 (1), 184-198 (2018).</li><li>Stobart, J. 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L., Holub, M., Weber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHIPS:+an+extensible+toolbox+for+cellular+and+hemodynamic+two-photon+image+analysis.">CHIPS: an extensible toolbox for cellular and hemodynamic two-photon image analysis.</a> Neuroinformatics. 16, 145-147 (2018).</li><li>Hall, C. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; of&#160; 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 &#34;image processing packages&#34;.&#160; 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 &#34;image processing algorithms&#34;. 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 &#34;image processor software&#34;.&#160; 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 &#34;programming platform &#34;. 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&#946;-CreERT2</td>
			<td>The Jackson Laboratory</td>
			<td>30201</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Tubing, PE10 I.D. 28mm (0.11&#8221;) O.D. 61mm (.024&#8221;)</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.5K Views.  University of Manitoba. 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.

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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 ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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 ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...