Distinct signatures of calcium activity in brain pericytes

Even though pericytes have been implicated in various neurological disorders, little is known about their function and signaling pathways in the healthy brain. Here, we characterized cortical pericyte calcium dynamics using two-photon imaging of Pdgfrβ-CreERT2;GCaMP6s mice under anesthesia in vivo and in brain slices ex vivo. We found distinct differences between pericyte subtypes in vivo: Ensheathing pericytes exhibited smooth muscle cell-like calcium dynamics, while calcium signals in capillary pericytes were irregular, higher in frequency and occurred in cellular microdomains. In contrast to ensheathing pericytes, capillary pericytes retained their spontaneous calcium signals during prolonged anesthesia and in the absence of blood flow ex vivo. Chemogenetic activation of neurons in vivo and acute increase of extracellular potassium in brain slices strongly decreased calcium activity in capillary pericytes. We propose that neuronal activity-induced elevations in extracellular potassium suppress calcium activity in capillary pericytes, likely mediated by Kir2.2 and KATP channel activation.


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The entire abluminal surface of the cerebral vasculature is covered by mural cells, namely 38 vascular smooth muscle cells (SMC) and pericytes, which exhibit a continuum of phenotypes. 39 Despite several attempts to categorize these heterogeneous cells in the adult mouse by  87 To study calcium dynamics in mural cells, we crossed Pdgfrβ-CreERT2 mice (Gerl et al.,88 2015)) with stop-flox GCaMP6s reporter mice ( Figure 1A). For localization and classification 89 of mural cells, we defined the vessel types based on their branch order and diameter. The were injected before the chronic cranial window implantation over the somatosensory cortex. Images 110

Two-photon imaging of Pdgfrβ-driven GCaMP6s in mural cells
were acquired at a wavelength of 940 nm and the vasculature was labeled via an intravenous injection 111 (iv.) of 2.5% Texas Red Dextran (70 kDa). In vivo experiments were conducted with anesthetized 112 (1.2% isoflurane) mice. For pharmacologic interventions, acute brain slices of the same mice were 113 prepared. Z-stacks of the vessel arbor were acquired to determine the precise location of the imaged 114 cells along the vasculature.  124 In all the mural cells described above we observed basal calcium fluctuations in somata (S) 125 and processes (P) in vivo (  To compare the calcium dynamics between EPs and CPs, we measured frequency, amplitude 134 and duration of calcium transients using semi-automated image analysis as described and  Figure 2B).

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Calcium signals in SMCs during spontaneous vasomotion are inversely correlated to vessel 148 diameter (transformed r Fisher z = 1.20 (0.37), n=13), as evaluated by cross-correlation analysis 149 ( Figure 2D), and reported in a previous study (Hill et al., 2015). We observed a similar   Persisting calcium signals in CPs ex vivo 195 To further investigate the differences in calcium signals between EPs and CPs, we prepared 196 acute brain slices for ex vivo pharmacological probing. Blood plasma was stained with an iv.  Figure 3A). In many cases ( Figure 3B), arteries 205 and arterioles were either collapsed due to loss of tone or intraluminal pressure in the slice 206 preparation or were constricted by possibly dead SMCs or EPs (no detectable calcium signal).
207 Surprisingly, CPs in ex vivo brain slices retained their highly frequent calcium transients, 208 which were also confined to microdomains ( Figure 3C). The comparison of basal calcium   The application of the thromboxane A2 receptor agonist U46619 (100 nM) to the slice 224 preparation is often used to prevent the collapse of wider calibre vessels by generating an 225 artificial vascular tone (Mishra et al., 2014, Brown et al., 2002. However, we observed a 226 massive calcium response in CPs when U46619 was included in the superfusate ( Figure 3D, 227 Video 5). This overt calcium response in CPs was accompanied by cytoplasmic extrusions, 228 suggesting that U46619 might be toxic for pericytes. In addition, blood plasma inside the 229 vessel was pushed away from the region where the CP soma was located ( Figure 3D).  branches of a pial artery lying on the surface of the brain slice. On the left, two-photon microscopy 237 image of a precapillary arteriole and its adjacent 1 st order branch, where an ensheathing pericyte is 238 located. The yellow box shows a magnified image of the ensheathing pericyte. In the GCaMP6s 239 channel, ROIs for soma (S, in magenta) and processes (P1-3, in blue) are shown. In the center are the 240 respective normalized traces of calcium signals. On the right are violin/box plots depicting the 241 quantified calcium signal frequency of ensheathing pericytes ex vivo compared to the previously 242 ( Figure  . The corresponding vessel image shows that blood plasma is moved away (white arrow 258 indicates the direction of movement) from the site where the soma is located in the center the 259 normalized trace of the calcium response of the whole cell is shown. On the right, quantification of the 260 area under the curve (auc) comparing baseline to U46619 treatment is shown. Data represents 261 individual cells, median and interquartile ranges. Statistics were calculated using a two-tailed 262 Wilcoxon matched-pairs signed rank test. N=5, n=16, P < 0.001. 263 Scale bars: 10 μm. n.s. indicates not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Data represents individual cells, median and interquartile ranges. Statistics were calculated using 299 Wilcoxon matched-pairs signed rank tests. Endothelin-1: N=3, n=7, P = 0.02; ATP: N=4, n=8, P = 300 0.008; UDP-Glucose: N=4, n=11, P < 0.001. 301 (D) Calcium response to application of the L-type voltage-gated calcium channel (L-type VGCC) 302 blocker Nimodipine (100 µM). On top is a representative normalized calcium signal trace and below is 303 the quantification of the signal frequency in somata and processes, comparing baseline to the 304 Nimodipine treatment. Nimodipine was infused 15 min prior to data collection. Data represents 305 individual cells, median and interquartile ranges. Statistics were calculated using two-tailed paired t-306 tests, N=4, n=11. S: t(10) = 2.39, P = 0.04; P: t(10) = 0.1495, P = 0.88. 307 (E) Calcium response to application of the TRPC channel blocker SKF96365 (100 µM). On top is a 308 representative normalized calcium signal trace and below is the quantification of the signal frequency 309 in somata and processes, comparing baseline to the SKF96365 treatment. Data represents individual 310 cells, median and interquartile ranges. Statistics were calculated using two-tailed paired t-tests, N=3, 311 n=6. S: t(5) = 4.038, P = 0.01; P: t(5) = 6.755, P = 0.001. 312 (F) Calcium response to application of TTX (1 µM). On top is a representative normalized calcium 313 signal trace and below is the quantification of the signal frequency in somata and processes, 314 comparing baseline to the TTX treatment. TTX was infused 5 min prior to data collection. Data 315 represents individual cells, median and interquartile ranges. Statistics were calculated using two-tailed 316 paired t-tests, N=3, n=9. S: t(8) = 1.113, P = 0.2979; P: t(8) = 0.5425, P = 0.6. 317 The red lines below the traces indicate the addition time of the respective drug. 318 n.s. indicates not significant, *P < 0.05, **P < 0.01,***P < 0.001.  Figure 5D).  Figure 5D).  Figure 5F). This response was 371 found to be specific for mural cells, since nearby GCaMP6s-labeled astrocytes reacted to the 372 hypoxic conditions with a massive calcium response ( Figure 5F). (on the left) shows a CP and neurons, expressing hM3D(Gq)-mCherry. A representative normalized 378 calcium signal trace is shown (in the center) and the calcium signal frequency is quantified, comparing 379 baseline to treatment (on the right). Data represents individual cells, median and interquartile ranges. 380 Statistics were calculated using a Wilcoxon matched-pairs signed rank test (for S) and a two-tailed 381 paired t-test (for P), N=4, n=12. S: P < 0.001; P: t(11) = 7.453, P < 0.001. Scale bar = 10 µm. pericyte; white arrowhead: astrocyte). Center, a representative normalized calcium signal trace is 414 shown. The calcium signal frequency is quantified on the right. Baseline is compared to hypoxic 415 intervention. Data represents individual cells, median and interquartile ranges. Statistics were 416 calculated using Wilcoxon matched-pairs signed rank tests, N=4, n=16. S: P < 0.001; P: P < 0.001. 417 The red lines indicate the addition time of the respective drug.   Another potent vasomodulator is potassium, which is released in high amounts during the 479 repolarization phase after action potential firing (Paulson and Newman, 1987). Potassium CPs could also provide a protective mechanism to manage over excitability and lowered 506 metabolic supply during hypoxia/ischemia, as has been proposed for a subset of neurons in 507 the brain (Ballanyi, 2004, Yamada andInagaki, 2005).

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Calcium is involved in a plethora of cellular processes, ranging from cellular homeostasis to 509 force generation (Berridge et al., 2003). This may explain the spread of calcium signals SMCs. The pericytic hyperpolarization inactivates TRPC and VGCC channels leading to a drop in 527 calcium signals. 528 Blockage of the respiratory metabolism by NaN3 or hypoxia leads to a presumed reduced 529 [ATP]/[ADP] ratio activating KATP channels to induce a hyperpolarization and a subsequent decrease 530 in calcium signals.     Pharmacology in brain slices 619 Slices were imaged at 34 °C in the same aCSF that was used for recovery after cutting.     applied to all images to reduce noise. A moving threshold for each pixel was defined in the 654 filtered stack as the mean intensity plus 7 times the standard deviation of the same pixel 655 during the preceding 2.46 s. Using this sliding box-car approach, active pixels were identified 656 as those that exceeded the threshold. Active pixels were grouped in space (radius = 2 µm) and 657 time (width = 1 s). Resulting ROIs with an area smaller than 4 µm 2 were considered to be 658 noise and were excluded. We then combined the previously hand-selected ROIs into a single 659 mask by subtracting the soma ROI from the whole cell territory ROIs, thereby leaving a mask 660 of pericytes without soma. We then multiplied this 2D mask with each frame of the 3D mask 661 obtained from the automated ROI detection in order to obtain a mask of ROIs within the cell 662 territory and outside of the soma. We then extracted traces from two sets of ROIs for each 663 image: the hand-selected soma ROIs and the adjusted 3D activity mask. The minimum 664 distance from each activity ROI to the nearest soma was defined as the shortest distance 665 between ROI edges. The signal vector (dF/F) from each ROI was calculated using the mean We thank the Viral Vector Facility of the University of Zürich for the supply of AAV vectors. 702 We are grateful to Marc Zünd for assembly and maintenance of two-photon microscopes.  Example image of ROI selection by hand in ImageJ. The soma was outlined and a border ROI was set 971 around the whole pericyte, so as to reduce detection of signals not associated with the observed cell. 972 To the right is a time overlay of all process ROIs found inside the border ROI. Scale bars: 10 μm.