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. 2013 May 8;33(19):8411-22.
doi: 10.1523/JNEUROSCI.3285-12.2013.

In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase

Krystal Nizar  1 , Hana Uhlirova, Peifang Tian, Payam A Saisan, Qun Cheng, Lidia Reznichenko, Kimberly L Weldy, Tyler C Steed, Vishnu B Sridhar, Christopher L MacDonald, Jianxia Cui, Sergey L Gratiy, Sava Sakadzić, David A Boas, Thomas I Beka, Gaute T Einevoll, Ju Chen, Eliezer Masliah, Anders M Dale, Gabriel A Silva, Anna Devor
Affiliations

In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase

Krystal Nizar et al. J Neurosci. .

Abstract

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knock-out (KO) mice, in which the primary mechanism of astrocytic calcium increase-the release of calcium from intracellular stores following activation of an IP3-dependent pathway-is lacking. Further, our results in wild-type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.

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Figures

Figure 1.
Figure 1.
Cross talk between astrocytic and neuropil calcium signals during stimulus-induced response. A, Reduction of cross talk with neuropil signals through shrinking (“erosion”) of astrocytic ROI in OGB1-stained cortex. An example FOV including a number of astrocytes labeled with SR101/OGB1 and a number of neuronal cell bodies labeled with OGB1, imaged 190 μm below the cortical surface. Scale bar, 20 μm. A gradual erosion of a single-cell ROI (astrocytic cell body labeled “a”) is illustrated on the right: Starting from a mask defined by a liberal thresholding of SR101 intensity (m1) and eroding a single layer of pixels at a time (m2-6). The masks are enlarged relative to the composite image for viewing purposes. B, Time courses extracted from the masks shown in A. All time courses are baseline-subtracted and peak-normalized to illustrate that the erosion procedure selectively decreases contamination from neuropil (black arrowheads) while preserving the slow and large-amplitude “true” astrocytic signal. Calcium signal change in neuropil (i.e., outside neuronal and astrocytic cell bodies), expressed as ΔF/F, is shown in green. The black bars indicate stimulus duration. C, An example FOV labeled with Fluo-4 and SR101, imaged 180 μm below the cortical surface. The yellow color of the composite image indicates colocalization of Fluo-4 (green) and SR101 (red) in astrocytes. Scale bars, 30 μm. D, Calcium signal time courses, expressed as ΔF/F, extracted from the ROIs outlined in the composite image in C. The black bars indicate stimulus duration. E, An example nonrectified MUA voltage trace illustrating responses to six consecutive stimuli within a single stimulus trial. MUA recordings were performed simultaneously with optical acquisition to ensure neuronal response to stimulation.
Figure 2.
Figure 2.
IP3R2-KO mice do not exhibit IP3-mediated calcium increases. A, Example response to microinjection of 1 mm ATP imaged 100 μm below the cortical surface in a WT subject. Left, A composite image of OGB1 (green), SR101 (red), and the injection micropipette (blue). Right, Astrocytic ROIs. Scale bar, 20 μm. B, Calcium signal time courses extracted from astrocytic ROIs in A. C, As in B for an IP3R2-KO subject. Every line represents a time course from an individual astrocytic cell body. Composite image and ROIs are not shown. D, Onsets of astrocytic calcium increases in response to ATP in WT subjects, as a function of distance from the injecting pipette. Astrocytes in IP3R2-KO subjects did not exhibit calcium increases and are not plotted. E, Example response to microinjection of 10 μm t-ACPD imaged 120 μm below the cortical surface in a WT subject: the first 3 puffs evoked time-locked calcium increases followed by irregular oscillations. F, As in E for an IP3R2-KO subject.
Figure 3.
Figure 3.
IP3R2-KO mice exhibit normal functional hyperemia. A, Time courses of arteriolar diameter change in WT and IP3R2-KO subjects (top and bottom panels, respectively). All measurements for each category are overlaid. The average is superimposed on each panel (thick lines). The stimulus onset is indicated by the gray vertical line. The across-subject averages are superimposed in the inset to facilitate temporal comparison; the error bars indicate a 95% confidence interval for the mean. B, Onset (top) and time-to-peak (bottom) for all measured arteriolar diameter changes in WT (solid dots) and IP3R2-KO (open triangles) subjects, extracted from the data in A. Data from all subjects are overlaid and presented as a function of the cortical depth. C, Comparison of paired diameter and velocity measurements from surface arterioles. Cross-subject averages for diameter (black) and velocity (green) time courses. First, we averaged all time courses acquired within a subject. Then, averaged time courses were normalized by the peak amplitude before calculating the average across subjects; the error bars indicate SE across subjects. An increase in velocity preceded an increase in diameter (p < 0.01). This behavior is consistent with a theoretical expectation from a distributed vascular network when the fastest dilation occurs in deep cortical layers (Boas et al., 2008). D, An example illustrating simultaneous measurements of diameter and velocity. On the left, a scan path is superimposed in red on an FITC image of a surface arteriole. A segment of the scan path along the vessel is used to estimate velocity based on the angle of streaks in temporally stacked lines denoted by α at the top right (see Materials and Methods) (Kleinfeld et al., 1998). A segment of the scan path across the vessel is used to compute dilation based on expansion of the profile (the red arrow at the top right). Corresponding diameter (black) and velocity (green) time courses are shown at the bottom right.
Figure 4.
Figure 4.
Robust arteriolar dilation is observed in the absence of an astrocytic calcium response. A, Representative FOV including a perivascular astrocyte labeled with SR101/OGB1, a diving arteriole labeled by intravascular injection of FITC, and a number of neuronal cell bodies labeled with OGB1, imaged 150 μm below the cortical surface. ROIs used for extraction of time courses are shown on the right. B, Time courses extracted from the ROIs shown in A. Astrocytic (red) and neuronal (green) calcium signal changes are expressed as ΔF/F. Vasodilation is expressed as percentage diameter change relative to the baseline diameter, Δd/d. The diameter change was extracted from the expansion of FITC-labeled intravascular lumen, indicated by “v” in A. The black bars indicate stimulus duration. C, Ratio images showing neuronal signal change and vasodilation (black arrowheads) in response to the first stimulus trial. The ROI contours are overlaid. Each image was computed as an average of five consecutive ratio frames. The corresponding time windows relative to the stimulus onset (in seconds) are indicated above the images. Note the 0.5 s gap between the consecutive images. Scale bars: A, C, 10 μm. D, “Raw” trial-averaged images of the upper part of the FOV illustrating the expansion of FITC-labeled cross-section during peak dilation (bottom) relative to the prestimulus baseline (top, the red dotted line). The green channel is shown. E, Representative example of a calcium signal time course extracted from a single neuronal cell body (black trace; labeled “n1” in B). Calcium signal changes are expressed as ΔF/F. A computational fit to the data is overlaid in red. The fitting procedure assumed a convolution kernel with τ = 0.8 s. The black bars at the bottom indicate stimulus duration. As a general rule, neurons fired 1–2 spikes in response to each of 6 electrical pulses in the stimulus train.
Figure 5.
Figure 5.
Astrocytes exhibit occasional calcium responses to individual stimulus trials. A, Example FOV 20 μm below an arteriolar branching point including three astrocytic ROIs, imaged 200 μm below the cortical surface. No intravascular FITC was present. B, Time courses extracted from astrocytic and neuronal ROIs shown in A. Red arrowheads point to a calcium increase in one of the ROIs (a1) during 2 of 10 stimulus trials. C, Trial-averaged ratio images for the first 1.8 s following the stimulus onset during the neuronal response. Every image is an average of two consecutive frames. Intensity fluctuations between consecutive images reflect temporally undersampled “flashing” of the neuropil and neuronal cell bodies in response to repetitive stimuli (6 individual stimuli at 3 Hz). The ROI contours from A are overlaid. The corresponding time windows relative to the stimulus onset (in seconds) are indicated above the images. D, Ratio images for the ninth stimulus trial featuring calcium response in the astrocytic ROI labeled a1. Time relative to the stimulus onset (in seconds) is indicated above the images. Note the 1 s gap between the consecutive images. Scale bars: A, C, D, 10 μm.
Figure 6.
Figure 6.
Astrocytic response kinetics as a function of the distance from arterioles. A, B, Overlaid time courses of all trials with significant astrocytic signal change, pooled across subjects, sorted by distance from diving arterioles: within (A) and outside (B) a 50 μm radius. C, Statistical comparison of astrocytic response kinetics for the two distance categories across subjects: <50 μm (red) and >50 μm (blue). The curves show across-subject averages; the error bars indicate SE.
Figure 7.
Figure 7.
Astrocytic calcium increase is delayed relative to arteriolar dilation. A, Overlaid time courses of all trials with significant astrocytic signal change, pooled across subjects with (left) and without (right) intravascular FITC used for simultaneous measurements of vasodilation. B, Onset (top) and time-to-peak (bottom) for all measured astrocytic calcium responses (red) and arteriolar diameter changes (black). Data from all subjects are overlaid and presented as a function of cortical depth. Open squares and solid dots correspond to simultaneous (st) and stand-alone (sa) measurements, respectively. Simultaneously measured diameters are included regardless of the presence of an astrocytic calcium response. C, Time courses of astrocytic calcium change (top) and arteriolar diameter change (bottom). An average is superimposed on each panel (thick lines). The stimulus onset is indicated by the gray vertical line. Peak-normalized averages are superimposed in the inset to facilitate temporal comparison.
Figure 8.
Figure 8.
Calcium increase in astrocytic endfeet lags behind dilation onset. A, An example FOV including two astrocytes, one (a1) with a connected endfoot (EF), imaged 260 μm below the cortical surface. B, Time courses extracted from ROIs shown in A. The red arrowhead points to a calcium increase in the endfoot in response to the seventh stimulus trial. C, Trial-averaged ratio images for the first ∼2.5 s following the stimulus onset during the neuronal response. Every image is an average of two consecutive frames. The ROI contours from A are overlaid. The corresponding time windows relative to the stimulus onset (in seconds) are indicated above the images. Note the 1 s gap between the consecutive images. D, Ratio images in response to the seventh stimulus trial. The ROI contours are overlaid. Each image was computed as an average of 10 consecutive ratio frames. The corresponding time windows relative to the stimulus onset are indicated above the images. Note the 1 s gap between the consecutive images. Scale bars: A, C, D, 10 μm. E, Overlaid time courses of astrocytic calcium change in the cell body (a1, black) and the connected endfoot (EF, red). F, All responsive endfoot trials are overlaid. The average is shown in thick red. The averaged dilation time course from the same depth category (180–300 μm), normalized to the maximum amplitude of the calcium traces, is superimposed in thick black.
Figure 9.
Figure 9.
Calcium increase in fine astrocytic arborizations lags behind dilation onset. A, An example FOV labeled with Fluo-4 and SR101, imaged 200 μm below the cortical surface. B, Subcellular ROIs overlaid on an MIP image of Fluo-4 ratio images. The MIP image was calculated for the time series of 3 stimulus trials. Individual ROIs were defined by thresholding MIP images calculated for individual stimulus trials. Scale bars: in A, B, 50 μm. C, Calcium signal time courses extracted from ROIs shown in B (red traces) and from the entire FOV (“Fluo-4 field,” blue trace). The black bars indicate stimulus duration. D, Averaged Fluo-4 field response. Averaged dilation time course and calcium response from astrocytic cell body ROIs from the same depth category (≤200 μm) are superimposed in black and red, respectively. All curves are normalized to the maximum amplitude.
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References

    1. Agulhon C, Petravicz J, McMullen AB, Sweger EJ, Minton SK, Taves SR, Casper KB, Fiacco TA, McCarthy KD. What is the role of astrocyte calcium in neurophysiology? Neuron. 2008;59:932–946. doi: 10.1016/j.neuron.2008.09.004. - DOI - PMC - PubMed
    1. Allaman I, Bélanger M, Magistretti PJ. Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci. 2011;34:76–87. doi: 10.1016/j.tins.2010.12.001. - DOI - PubMed
    1. Anderson LA, Christianson GB, Linden JF. Mouse auditory cortex differs from visual and somatosensory cortices in the laminar distribution of cytochrome oxidase and acetylcholinesterase. Brain Res. 2009;1252:130–142. doi: 10.1016/j.brainres.2008.11.037. - DOI - PubMed
    1. Attwell D, Buchan AM, Charpak S, Lauritzen M, Macvicar BA, Newman EA. Glial and neuronal control of brain blood flow. Nature. 2010;468:232–243. doi: 10.1038/nature09613. - DOI - PMC - PubMed
    1. Bergles DE, Jahr CE. Synaptic activation of glutamate transporters in hippocampal astrocytes. Neuron. 1997;19:1297–1308. doi: 10.1016/S0896-6273(00)80420-1. - DOI - PubMed

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