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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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/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 ...

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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, ...

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 ...

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 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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...