An artery-specific fluorescent dye for studying neurovascular coupling

Journal name:
Nature Methods
Volume:
9,
Pages:
273–276
Year published:
DOI:
doi:10.1038/nmeth.1857
Received
Accepted
Published online

We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus–evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments.

At a glance

Figures

  1. Selective labeling of artery walls by Alexa Fluor 633.
    Figure 1: Selective labeling of artery walls by Alexa Fluor 633.

    (a,b) Two-photon microscopy image of the mouse visual cortex after a 4 pounds per square inch (p.s.i.) 1-s puff of Alexa Fluor 633 from a micropipette (a; + indicates tip position) and an autofluorescence image (b) obtained simultaneously in vivo using 2 mW laser power. (c,d) Image from the rat visual cortex after a 7 p.s.i. 1-s puff of Alexa Fluor 633 in vivo (c) and fluorescence image of the same cortical site taken after intravenous injection of fluorescein dextran (d). (e,f) Images of arteriole walls in mouse visual cortex after intravenous injection of Alexa Fluor 633 (e) and of the same site after injection of fluorescein dextran (f). (g) Average intensity 66 μm z-dimension projection image from mouse visual cortex after an intravenous injection of fluorescein dextran and local pipette injection of Alexa Fluor 633. (h) Dilation of an Alexa Fluor 633–labeled arteriole from the rat visual cortex in response to drifting grating visual stimuli (gray bar). The intersection between the regression line (dotted green) and zero level (dashed black) represents the latency of dilation (0.8 s). Purple shading indicates s.e.m. (n = 16 trials). (i) Molecular structures of Alexa Fluors 633 and 594. (j) Relative brightness of arteriole walls labeled with Alexa Fluors 594, 633 and 647. Data were pooled from five experiments in the rat cortex (error bars, s.e.m.). (k) Image of an immunostained section from the macaque monkey neocortex using an antibody to α-SMA and showing Alexa Fluor 633 labeling. (l) Image of an immunostained section of mouse neocortex with an antibody to GLUT1 and showing Alexa Fluor 633 labeling. (m) Image of a mouse aorta labeled with Alexa Fluor 633 and collected by using 2 mW two-photon laser power. (n) Image of a mouse femoral artery stained with an antibody to laminin and showing Alexa Fluor 633 labeling. In vivo images (af) were not background-subtracted or movement-corrected. Images shown in k, l and n were collected with sequential scanning confocal microscopy. Scale bars, 100 μm (ag), 10 μm (k,l,n) and 50 μm (m).

  2. Monitoring sensory stimulus-evoked responses in arterioles and adjacent neurons.
    Figure 2: Monitoring sensory stimulus–evoked responses in arterioles and adjacent neurons.

    (ad) Simultaneous tracking of neuronal activation using the calcium dye Oregon Green 488 Bapta-1 acetoxymethyl ester (OGB-1 AM) and changes in arteriole diameter using Alexa Fluor 633 in layer 2/3 of the cat visual cortex. Image of an Alexa Fluor 633–labeled (magenta) penetrating arteriole and adjacent neuronal cell bodies labeled with OGB-1 AM (green) (a). Scale bar, 50 μm. Time courses (averages of 24 trials) of responses to drifting grating visual stimuli from neurons (b,c) and arteriole (d) marked in a. ΔF/F, relative change in fluorescence. (eg) Measurement of red blood cell velocity and vessel diameter in an arteriole of the rat visual cortex. Imaged field of view (e) showing an arteriole labeled with Alexa Fluor 633 (magenta) and its lumen labeled with fluorescein dextran (green). Scale bar, 20 μm. Time courses (averages of 32 trials) of increases in velocity (f) and diameter (g) in response to drifting grating visual stimuli measured at locations marked by dotted lines in e. (h,i) Dilation of an Alexa Fluor 633–labeled arteriole in the cat visual cortex (h) in response to the presentation of visual stimuli in each of the nine locations (3 × 3 grid) of a stimulus display monitor (i). Average responses to five repeats of the entire visual stimulus sequence are plotted in h. The response to each stimulus position is plotted in the corresponding location in the 3 × 3 grid in which the luminance value represents the dilation magnitude. All gray bars, period of visual stimulation; error bands, s.e.m.

  3. Stimulus-dependent modulation of fluorescence in neurons located under cortical surface arterioles but not venules.
    Figure 3: Stimulus-dependent modulation of fluorescence in neurons located under cortical surface arterioles but not venules.

    (ad) Mask representations of the dorsal view of a surface arteriole (a), venule (c) and underlying neuronal soma (labeled 1–4) in the rat visual cortex, and Alexa Fluor 594 fluorescence in the indicated neurons after a visual stimulus, in the same rat (b,d; averages of 23 trials). (e) Rendering of a surface arteriole (magenta) and a neuronal soma (green) in an imaged cortical volume, illustrating the potential dependence of the neuronal imaging artifact on the relative positioning of an arteriole and a nearby neuron. The two paths of light were achieved by rotating the head of a mouse that was positioned under the two-photon microscope's objective lens. In head angle 1, light passes through the surface arteriole. In head angle 2, light beam bypasses the arteriole. (f) Mask representation of an imaged area in mouse visual cortex (dorsal view) showing a surface arteriole (magenta) and a neuronal soma located directly below. (g) Alexa Fluor 594 fluorescence from the soma shown in f in response to visual stimuli. (h) Dorsal view of the same area as in f but after head rotation; soma is no longer under the arteriole. (i) Alexa Fluor 594 fluorescence in the soma shown in h in response to visual stimuli. Time courses in g and i are averages of 31 trials. All gray bars represent the period of visual stimulation; error bands, s.e.m.; scale bars, 50 μm.

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Author information

  1. These authors contributed equally to this work.

    • Zhiming Shen &
    • Zhongyang Lu

Affiliations

  1. Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA.

    • Zhiming Shen,
    • Zhongyang Lu,
    • Pratik Y Chhatbar,
    • Philip O'Herron &
    • Prakash Kara

Contributions

P.K. conceived and designed the study, except the head angle with dip artifact experiments (Z.S.). All authors performed in vivo vessel imaging experiments. Z.S. did in vivo calcium imaging and head-angle experiments. Z.L. did histology on brain and femoral vessel tissue samples. P.K. did histology of mouse aorta, mouse kidney and human aorta tissues. P.K., Z.S., P.Y.C. and P.O. analyzed the in vivo vessel dilation and velocity data. P.K. and Z.S. analyzed the in vivo calcium imaging and single-cell electroporation data. All authors discussed results. P.K. wrote the manuscript.

Competing financial interests

The Medical University of South Carolina Foundation for Research Development has filed a provisional patent application US 61/626,314 that names P.K. as an inventor and encompasses aspects of the technology described in this paper.

Corresponding author

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Supplementary information

PDF files

  1. Supplementary Text and Figures (10M)

    Supplementary Figures 1–20, Supplementary Notes 1–2

Movies

  1. Supplementary Video 1 (1M)

    Delay between the filling of arteries and veins after intravenous dye injection confirm artery specificity of Alexa Fluor 633 labeling in vivo. Time-lapse epifluorescence imaging of cortical vessels immediately after fluorescein dextran was injected intravenously in the femoral vein. The entire x-y field of view shown represents a region of 1.87 mm × 1.87 mm in the cat visual cortex. Because the dye must pass from the femoral (leg) vein through the heart to get to the cerebral cortex, the arteries must label first. Images were collected using a monochrome CCD camera (see Supplementary Fig. 1).

  2. Supplementary Video 2 (3M)

    Small cortical surface arterioles were labeled in vivo by Alexa Fluor 633, whereas venules were not. High-optical-zoom two-photon z stack from the rat visual cortex. The x-y field of view shown represents a region of 109 μm × 109 μm. The 18 z steps of the movie span 34 μm. The lumen of the vessels is visible (green) after an intravenous injection of fluorescein dextran. The vessel walls of a primary arteriole and daughter branchlet are well labeled by an intravenous injection of Alexa Fluor 633 (red) whereas a nearby venule is unlabeled (see Supplementary Fig. 2).

  3. Supplementary Video 3 (8M)

    Cortical surface and penetrating arteriole ≥15 μm diameter were labeled in vivo by Alexa Fluor 633 whereas veins and microvessels were not. A 360° rotation view of a 220 μm × 234 μm × 123 μm two-photon volume from mouse visual cortex. All vessels are visible (green) after an intravenous injection of fluorescein dextran but only the surface and penetrating arterioles are labeled by Alexa Fluor 633 (Supplementary Fig. 5c,d).

  4. Supplementary Video 4 (315K)

    Raw two-photon imaging frames showing an increase in arteriole vessel diameter upon sensory visual stimulation in vivo. Raw data frames (left) and frame-locked presentation (right) of blank (gray) and drifting grating visual stimuli. Data are shown for 15 frames: 5 frames blank, followed by 5 frames of visual stimulation and then 5 frames blank. The duration of each frame was 1.48 s. The entire x-y field of view shown in the 2 two-photon imaging frames represents a region of 553 μm × 553 μm from rat visual cortex close to the pial surface.

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