Orai, RyR, and IP3R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Phillips, Braxton; Clark, Jenna; Martineau, Éric; Rungta, Ravi L.

doi:10.1038/s42003-023-04858-3

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Published: 06 May 2023

Orai, RyR, and IP₃R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Communications Biology volume 6, Article number: 493 (2023) Cite this article

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Abstract

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca²⁺ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca²⁺ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca²⁺ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca²⁺ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca²⁺ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca²⁺ transients in mid-capillary pericytes occur through a combination of IP₃R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca²⁺ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca²⁺ influx via Orai channels reciprocally regulates IP₃R and RyR release pathways in the ER, which together generate spontaneous Ca²⁺ transients and amplify Gq-coupled Ca²⁺ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca²⁺ and a target for manipulating their function in health and disease.

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Introduction

The mural cells of the brain are multifunctional cells organized in a continuum along the arterio-venous axis of the cerebral vasculature. This continuum is grouped into distinct cell types, which includes smooth muscle cells on penetrating arterioles (aSMCs), pre-capillary sphincters and ensheathing pericytes (also named terminal SMCs) of the arteriole-to-capillary transition zone, pericytes of the mid-capillary bed (of mesh and thin-strand morphology – here collectively referred to as mid-capillary pericytes), and venule smooth muscle cells^1,2. Although they share a common origin³ and similar nomenclature, these cell types have numerous morphological^4,5, transcriptomic^6,7, and functional differences^8,9. For example, aSMCs and ensheathing pericytes fully encircle and exhibit near complete coverage of the endothelial tube, highly express alpha smooth muscle actin (α-SMA), and undoubtedly regulate cerebral blood flow (CBF)^9,10. Mid-capillary pericytes have thin or mesh-like processes that run longitudinal to the vessel, express little to no α-SMA, and their role in controlling CBF in physiological contexts remains poorly defined. Nevertheless, blood flow control aside, mid-capillary pericytes have numerous functions in health and disease, such as blood–brain-barrier regulation^11,12,13 neuroimmune regulation¹⁴, angiogenesis¹⁵, glial scar formation¹⁶, and suggested stem cell-like properties¹⁷. Yet, despite these important functions, the basic physiological properties of brain mid-capillary pericytes, including the mechanisms that regulate their Ca²⁺ signaling, remain understudied.

All mural cells exhibit spontaneous fluctuations in intracellular Ca²⁺, which are termed Ca²⁺ transients^9,18,19. Whereas the mechanisms underlying calcium signaling and contraction in aSMCs have been studied in detail (e.g. refs. ^{20,21,22,23,24,25,26}.), investigations into brain pericyte Ca²⁺ signaling mechanism are only beginning to emerge. In ensheathing-type pericytes, Ca2+ transients can be caused by transmembrane influx through voltage-gated Ca2+ channels (VGCCs) and inositol 1,4,5-trisphosphate receptor (IP3R) signaling that recruits the Ca2+ required for α-SMA-mediated contraction^27,28. Mid-capillary pericytes also exhibit Ca²⁺ transients with similar properties in vivo and in vitro, which are confined to spatial microdomains and modulated by neuronal activity^9,18,29. Interestingly, a recent study reported that in resting conditions mid-capillary pericyte Ca²⁺ transients were only minimally sensitive to the potent L-type VGCC blocker nimodipine but were largely inhibited by the non-selective ion channel blocker SKF-96365¹⁸, consistent with a prior study reporting reduced functional expression of VGCCs on distal retinal capillaries³⁰. In addition to non-selective cation TRPC channels, SKF-96365 blocks several other Ca²⁺ channels that are highly expressed in mid-capillary pericytes such as T-type VGCCs³¹, and the Orai family of store-operated Ca²⁺ channels³². Mid-capillary pericytes also express a plethora of Gq-coupled receptors^7,33, which when activated would be expected to trigger Ca²⁺ release from internal stores via IP₃R signaling. Therefore, a more detailed investigation is required to understand the cellular mechanisms underlying Ca²⁺ signaling in mid-capillary pericytes, their voltage dependence, and the interplay between Ca²⁺ release from internal stores and transmembrane influx.

Here, we utilized pharmacology experiments on acute cortical brain slices from transgenic mice expressing a fluorescent calcium indicator, GCaMP6f, in brain mural cells expressing PDGFRβ (PDGFRβ-Cre;GCaMP6f mice) to delineate the mechanisms underlying Ca²⁺ signaling in brain pericytes. Surprisingly, in contrast to ensheathing type pericytes which we show exhibit Ca²⁺ transients that are partially dependent on VGCCs, mid-capillary pericyte Ca²⁺ signals are largely unaffected by inhibition of VGCCs. We show that mid-capillary pericyte Ca²⁺ transients are, instead, primarily mediated by an interplay between store-operated Ca²⁺ entry (SOCE) channels and release of Ca²⁺ from internal stores by IP₃Rs and ryanodine receptors (RyRs). Underscoring the potential importance of pericyte SOCE channels, we further demonstrate that they amplify Gq-coupled [Ca²⁺]_i elevations evoked by the potent vasoconstrictor endothelin-1 (ET-1).

Results

Pericyte Ca²⁺ signals were visualized with confocal imaging in acute cortical slices from PDGFRβ-Cre;GCaMP6f mice. The capillary lumen was labeled with an intravenous (I.V.) injection of RhodamineB-Dextran (70 kDa) and pericyte somas were labeled with the recently described pericyte specific dye TO-PRO-3³⁴ (Fig. 1a). The spontaneous and spatially incoherent nature of mid-capillary pericyte Ca²⁺ transients led us to take advantage of the recently developed event-based analysis tool AQuA, which was developed precisely for unbiased analysis of such signals, but in astrocytes³⁵. When applied to pericytes, AQuA was found suitable for extracting several properties of these pericyte Ca²⁺ transients, such as overall event frequency, amplitude, area, and duration (Fig. S1a–c; Supplementary Movie 1; and Tables S1 and S2). Indeed, Ca²⁺ transient frequency in mid-capillary pericyte somas as measured by AQuA was well-matched to measurements made with a region-of-interest based approach (Fig. 1b–d and S1d).

**Fig. 1: Imaging of mid-capillary pericyte Ca²⁺ transients and their dependence on extracellular Ca²⁺.**

We first tested whether these Ca²⁺ transients depended on extracellular Ca²⁺ ([Ca²⁺]_ex) by washing out [Ca²⁺]_ex from the perfused artificial cerebral spinal fluid (ACSF). Perfusion of 0 mM Ca²⁺, 2 mM EGTA solution reversibly depressed the frequency of Ca²⁺ transients in both the processes and soma of the pericyte (Fig. 1b–e and S1b–d). The inhibition of Ca²⁺ transients by Ca²⁺-free solution was rapid, reaching a maximal effect within 5–9 min (Fig. S1e). To test whether intracellular Ca²⁺ stores were completely depleted following 10 min in Ca²⁺-free ACSF, we applied Gq-coupled GPCR agonists for either the endothelin (ET)-A or thromboxane A2 receptor, ET-1 and U46619 respectively, which increase intracellular pericyte Ca²⁺ in an IP₃-dependent manner. ET-1 and U46619 were still able to evoke Ca²⁺ transients in Ca²⁺-free solution (Fig. 1f, g), suggesting that Ca²⁺ stores were not fully depleted at this time point. However, after prolonged exposure to Ca²⁺ free solution (30 min) signals generated by ET-1 application were severely blunted when compared to those induced at 5 and 10 min following [Ca²⁺]_ex removal (Fig. S2), suggesting a gradual run down of store Ca²⁺ levels in the absence of transmembrane Ca²⁺ influx, and a dependence on [Ca²⁺]_ex to maintain ER store Ca²⁺. Intriguingly, in Ca²⁺ free solution the ET-1 and U46619 induced increases in Ca²⁺ transient frequency were temporary (ET-1, 248.2 ± 46.52 s; U46619, 268.6 ± 32.65 s) (Fig. S2a) in contrast to the sustained global elevation previously observed in normal [Ca²⁺]_ex solution¹⁸. Altogether, these results suggest mid-capillary pericyte Ca²⁺ transients require a plasmalemmal influx pathway, either to directly generate the transients or to sustain constitutive intracellular store filling and release.

In ensheathing pericytes, depolarization triggers transmembrane influx of Ca2+ via VGCCs, thereby increasing Ca2+ transient frequency and [Ca²⁺]_i^27,28. Therefore, we tested the effect of blocking the predominantly expressed pericyte VGCC subtypes, Ca_V1.2 (L-type) and Ca_V3.2 (T-type), with nifedipine and Z944³⁶, respectively (Fig. 2a). Application of nifedipine (20 µM) and Z944 (2 µM) had no effect on the frequency of Ca²⁺ transients, or baseline Ca²⁺ levels, in either the processes or soma (Fig. 2b, S4, and Tables S1 and S2). However, we could not exclude the possibility that VGCC transients would become more apparent if the membrane were depolarized, as previously reported for ensheathing type pericytes²⁷. Consistent with this previous report, Ca²⁺ transient frequency of ensheathing pericytes was robustly elevated by increasing extracellular K⁺ concentration from 2.5 mM to 60 mM and was significantly reduced in the presence of VGCC blockers (Fig. 2c–g and Tables S1 and S2, 1st to 3rd branch order from penetrating arteriole). In contrast, depolarization with 60 mM [K⁺]_ex did not increase mid-capillary pericyte Ca²⁺ transient frequency (Fig. 2d) and VGCC blockers still had no effect on mid-capillary pericyte Ca²⁺ transient frequency when applied in 60 mM [K⁺]_ex (Fig. 2c–g and Tables S1 and S2). To further test the effect of membrane voltage on mid-capillary pericyte Ca²⁺, we pharmacologically altered K_ATP channel activity, whose modulation has been shown to have large effects on pericyte membrane potential^37,38,39. Consistent with our above results showing that mid-capillary Ca²⁺ transients were independent of VGCCs, application of pinacidil (10 µM), a K_ATP channel opener which hyperpolarizes pericytes, had no effect on transient frequency in processes or the soma (Fig. 2h). Furthermore, glibenclamide (20 µM) a K_ATP channel blocker, decreased rather than increased transient frequency in mid-capillary pericyte processes (Fig. 2i), suggesting that mid-capillary K_ATP channels were open at rest, and that depolarization may actually decrease Ca²⁺ influx. As a note, all experiments were performed in tetrodotoxin (TTX, 500 nM) to block neuronal action potentials, which alone had no significant effect on the frequency of mid-capillary pericyte Ca²⁺ transients, consistent with a previous report¹⁸, although resting [Ca²⁺]_i was slightly increased in TTX (Figs. S3 and S4 and Tables S1 and S2). Taken together, these results indicate that in contrast to ensheathing pericytes which exhibit elevated Ca²⁺ transients upon depolarization via VGCCs, mid-capillary pericyte Ca²⁺ transients are largely independent of VGCCs.

**Fig. 2: Mid-capillary pericyte Ca²⁺ transients are not potentiated by depolarization or mediated by VGCCs.**

Having established a marked difference in the mechanisms of Ca²⁺ signaling between pericyte subtypes, we next examined the unidentified calcium influx pathway(s) required for the generation of Ca²⁺ transients in mid-capillary pericytes (Fig. 3a). We first tested the non-specific ion channel blocker SKF-96365, which was previously reported to block mid-capillary pericyte Ca²⁺ transients¹⁸. Consistent with this previous report, Ca²⁺ transient frequency in the processes and somas of these pericytes was largely diminished in the presence of SKF-96365 (100 µM) (Fig. 3b and Tables S1 and S2). As SKF-96365 is a potent TRPC channel blocker, and TRPC3/6 are non-selective cation channels highly expressed in pericytes⁷, we further examined the sensitivity of mid-capillary pericyte Ca²⁺ transients to the TRPC3 blocker, Pyr3 and the potent TRPC3/6 blocker GSK-2833503A. Mid-capillary pericyte Ca²⁺ transients were unaffected by TRPC3 inhibition with Pyr3 (20 µM), and inhibition of TRPC3/6 with GSK-2833503A (10 µM) (Fig. 3b and Tables S1 and S2), suggesting influx via another SKF-96365 sensitive pathway. Interestingly, mid-capillary pericytes highly express Orai1 and Orai3 Ca²⁺ channels⁷, and these channels are also sensitive to SKF-96365³². We therefore tested the sensitivity of mid-capillary pericyte Ca²⁺ transients to the non-selective Orai channel blocker 2-APB. 2-APB is a potent Orai1 channel blocker at high concentration but potentiates Orai1 channels at low concentrations⁴⁰. Consistent with the bidirectional concentration-dependent sensitivity of Orai1 channels to 2-APB, 10 µM 2-APB increased mid-capillary pericyte Ca²⁺ transient frequency, whereas 100 µM 2-APB nearly abolished all transients (Fig. 3c and Tables S1 and S2). Although the effects of 2-APB are supportive of Orai mediated Ca²⁺ entry, 2-APB has several off-target effects, such as inhibiting IP₃Rs and some TRP channels. Therefore, we tested the sensitivity of mid-capillary pericyte Ca²⁺ transients to the Orai-specific blocker GSK-7975A⁴¹. Indeed, GSK-7975A (40 µM) robustly reduced the frequency of Ca²⁺ transients in mid-capillary pericytes in both the soma and processes (Fig. 3d–g and Tables S1 and S2). Additionally, we tested the effects of the recently developed compound IA65, which enhances Orai1/3 but inhibits Orai2^42,43. Consistent with the high mRNA expression of Orai1 and Orai3 in mid-capillary pericytes, IA65 (10 µM) rapidly induced an elevation in resting Ca²⁺ levels (Fig. 3h and S4), although in contrast to 2-APB (10 µM), we did not observe an increase in Ca²⁺ transient frequency (Tables S1 and S2).

**Fig. 3: Mid-capillary pericyte Ca²⁺ transients signals are dependent on Orai Ca²⁺ channels.**

While our data suggests that Ca²⁺ influx through Orai SOCE channels is required for the generation of spontaneous Ca²⁺ transients in mid-capillary pericytes, store-operated entry and store-release mechanisms are inextricably linked. Therefore, we set out to test the dependence of mid-capillary pericyte Ca²⁺ transients on store Ca²⁺ filling and release pathways (Fig. 4a). If mid-capillary pericyte Ca²⁺ transients were dependent on store release, then blocking store filling would be expected to block the generation of the transients. Indeed, pharmacological inhibition of the sarco(endo)-plasmic reticulum calcium-ATPase (SERCA), with cyclopiazonic acid (CPA, 30 µM) abolished Ca²⁺ transients (Fig. 4b) suggesting their dependence on store release. In parallel to the drop in transient frequency, CPA induced a global and sustained resting Ca²⁺ elevation (Fig. 4b), consistent with the activation of Orai channels via ER Ca²⁺ depletion and STIM1/2 proteins. Given the dependence of the Ca²⁺ transients on store Ca²⁺, we proceeded to investigate their dependence on the ER store release channels, IP₃Rs and RyRs. To test the relative contributions of these channels to mid-capillary pericyte Ca²⁺ transients, we either inhibited phospholipase C with U73122 (which prevents IP₃ production) or RyRs with the reversible antagonist tetracaine. Blocking IP₃ production with U73122 (25 µM) led a modest reduction in Ca²⁺ transient frequency in mid-capillary pericyte processes but not soma (Fig. 4c, d), and the spatial area of the remaining transients was also reduced (Fig. 4e). Strikingly, blocking RyRs with tetracaine (200 µM) robustly reduced the frequency, spatial area, and duration of Ca²⁺ transients in mid-capillary pericyte processes and soma (Fig. 4g–j and Tables S1 and S2), as well as resting Ca²⁺ levels (Fig. S4). We additionally confirmed the reversibility of the tetracaine effect on transient frequency in five cells (200–500 µM tetracaine = 30.33% ± 8.610%, washout = 108.2% ± 25.01% (% baseline frequency, mean ± SEM); p = 0.0305, two-tailed paired Students’ t test). Consistent with a functional role of RyRs in mid-capillary pericytes, application of caffeine (10 mM), a RyR potentiator, elevated mid-capillary pericyte Ca²⁺ (Fig. 4f). Altogether, these results point to a mechanism whereby constitutive Ca²⁺ entry through SOCE channels is required to maintain spontaneous Ca²⁺ release through RyRs and IP₃Rs, which in turn lead to store depletion and activation of Orai channels.

**Fig. 4: Mid-capillary pericyte Ca²⁺ transients are mediated by RyR and IP₃R store release pathways.**

To confirm that the Ca²⁺ influx through Orai channels was indeed store-operated, we tested whether Ca²⁺ entry to maximal store depletion was also sensitive to Orai channel antagonists, by adapting a protocol commonly used to maximally activate SOCE in expression systems and cell culture⁴⁴. First, we applied CPA (30 µM) for 20 min in Ca²⁺-free ACSF to deplete ER stores and maximally activate “store-operated” channels. Then, we reintroduced 1.2 mM [Ca²⁺]_ex to the bathing solution while imaging a mid-capillary pericyte (Fig. 5a, b), triggering a large influx of Ca²⁺ through the open transmembrane channels. As expected, a large increase in mid-capillary pericyte Ca²⁺ was measured upon reintroduction of [Ca²⁺]_ex, indicative of functional store-operated Ca²⁺ channels (Fig. 5a–c). As with the Ca²⁺ transients, this store depletion-induced Ca²⁺ influx was also sensitive to the Orai channel blockers, 2-APB and GSK-7975A, in mid-capillary pericytes (Fig. 5a, c). However, this experiment alone does not prove that store Ca²⁺ levels dictate the opening of the plasma membrane Orai channels, as the increase in cytoplasmic Ca²⁺ upon reintroduction of [Ca²⁺]_ex could simply be occurring through store independent channels that are constitutively open. Therefore, we performed an experiment in which the brain slice was exposed to Ca²⁺-free solution for the same duration as in the CPA experiments outlined above, but in the presence of tetracaine and U73122 to block store release via RyRs and IP3Rs. If plasma membrane Ca²⁺ influx is truly controlled by ER store Ca²⁺ levels in mid-capillary pericytes, then the increase in [Ca²⁺]_i upon reintroduction of [Ca²⁺]_ex should be smaller than when stores are depleted and SOCE is activated. Indeed, in slices perfused with 200 µM tetracaine and 25 µM U73122, re-introduction of [Ca²⁺]_ex resulted in a minute increase in Ca²⁺ entry that was not significantly different than when stores were depleted in the presence of Orai inhibitors (Fig. 5a, c). These results confirm that plasma membrane Ca²⁺ influx in mid-capillary pericytes is driven by ER store depletion.

**Fig. 5: Mid-capillary pericytes express functional SOCE, that is blocked with Orai inhibitors.**

The above evidence supporting SOCE channels in mid-capillary pericytes raised the intriguing possibility that Orai channels may be required to amplify and sustain GPCR mediated Ca²⁺ elevations, following robust release of Ca²⁺ from internal stores, as has been reported in other cell types (e.g. ref. ⁴⁵). Therefore, we harnessed the potent vasoconstrictor ET-1, which has been previously shown to induce large elevations in mid-capillary pericyte Ca²⁺¹⁸. Indeed, in 1.2 mM [Ca²⁺]_ex, ET-1 triggered a robust and sustained, but highly variable, Ca²⁺ elevation in mid-capillary pericytes (Fig. 6a, b). In contrast to the previously described role of the Ca²⁺ activated chloride channel, TMEM16A, and VGCCs, which are required to amplify ET-1 mediated Ca²⁺ elevations in ensheathing type pericytes (1st–3rd branch orders)²⁸, in mid-capillary pericytes, blocking VGCCs (20 µM nifedipine and 2 µM Z944) or TMEM16A (2 µM Ani9) for 20 min had no significant effect on the magnitude of the ET-1 mediated Ca²⁺ elevation (Fig. 6a, b). However, when Orai channels were blocked with GSK-7975A for 20 min prior to ET-1 application, the magnitude of this elevation in [Ca²⁺]_i was strongly reduced (Fig. 6a, b), and as in Ca²⁺-free ACSF (Fig. S2), ET-1 evoked a transient increase in event frequency rather than a sustained elevation (Fig. 6c, d). Importantly, these Ca²⁺ elevations were prevented by U73122 (25 µM, 20 min), confirming that ET-1 induced signals are initiated by the Gq-GPCR-PLC pathway (Fig. 6a, b). These results suggest that Orai mediated SOCE amplifies Gq-GPCR mediated Ca²⁺ elevations and may contribute to sustained increases in mid-capillary pericyte Ca²⁺ following the release of vasoconstrictive agents.

**Fig. 6: Ca²⁺ influx via Orai SOCE is required to sustain and amplify endothelin-1 induced [Ca²⁺]_i elevations.**

Discussion

Here we demonstrate that spontaneous Ca²⁺ transients in mid-capillary brain pericytes are mediated by an interplay between plasma membrane SOCE channels and ER store release through RyRs and IP₃Rs. Our results are suggestive of bidirectional coupling, whereby Orai transmembrane Ca²⁺ influx is required to sustain spontaneous Ca²⁺ release through RyRs and IP₃Rs, which in turn lead to store depletion and activation of Orai SOCE. These mechanisms are difficult to study in isolation: SOCE is dependent on store depletion, while store release is dependent on ER luminal Ca²⁺ load and intracellular Ca²⁺ levels, which are both heavily dictated by SOCE channels. Nevertheless, our pharmacological evidence, in conjunction with the short duration (~1–3 s) and spatially propagating nature of the observed Ca²⁺ transients, are consistent with a model in which spontaneous store release through RyRs and IP₃Rs is maintained by constitutive SOCE^46,47. Orai channels are ideally suited due to their high Ca²⁺ selectivity and tight coupling with luminal [Ca²⁺] in the ER via STIM proteins. Orai Ca²⁺ influx may additionally be critical for RyR activation by recruiting the intracellular Ca²⁺ required for calcium-induced-calcium-release (CICR) (e.g., ⁴⁸), a mechanism which classically depends on L-type VGCCs in cardiac muscle⁴⁹ and in smooth muscle²¹.

Our study adds to a growing body of evidence that the mural cells of the cerebral vasculature represent a continuum, with morphological, molecular, and functional heterogeneity along the arterio-venous axis. Our work is largely in agreement with a previous study which found that Ca²⁺ transients in mid-capillary pericyte processes were independent of L-type VGCCs at rest. However, in slight contrast to our results with 20 µM nifedipine, a minimal decrease in Ca²⁺ transient frequency within the soma was reported with 100 µM nimodipine¹⁸. Here we extend on these results and further show that these transients are not increased by depolarization and are likewise also independent of low-voltage-activated T-type calcium channels (Ca_V3.2), which pericytes robustly express⁷. These findings contrast with our initial hypothesis that these transients would require VGCC activity, which was based on our previous finding that [Ca²⁺]_i and Ca²⁺ transient frequency decreases in mid-capillary olfactory bulb pericytes following local increases in neuronal activity, although on a slower time scale than upstream ensheathing pericytes and SMCs⁹. While we show that VGCCs are not required for mid-capillary pericyte Ca²⁺ transients, they highly express both L- and T-type VGCCs⁷ and it is possible that these channels become inactivated at ~−20 mV when exposed to 60 mM [K⁺]_ex. Therefore, we cannot conclude that these channels are entirely functionally absent, but rather that Ca²⁺ signaling in mid-capillary pericytes is dominated by other mechanisms, which we report here. Intriguingly, a concurrent study has reported that distal retinal pericytes do show nifedipine-sensitive Ca²⁺ elevations in response to 60 mM [K⁺]_ex⁵⁰. However, in contrast to SMCs and ensheathing pericytes, L-type VGCCs were not required for the Ca²⁺ elevation in retinal mid-capillary pericytes following increased intraluminal pressure⁵⁰, consistent with our results exhibiting that Ca²⁺ transients in different subtypes of pericytes exhibit a differential dependence on VGCCs.

Our results in ensheathing pericytes are largely consistent with recent reports that have uncovered prominent roles of depolarization, L-type VGCCs, and Gq-GPCR-IP₃R signaling in mediating their Ca²⁺ transients and [Ca²⁺]_i^27,28. Elucidating the molecular and functional differences between pericyte subtypes, including in the Ca²⁺ signaling mechanisms described here, remains an important task. While SOCE is universal across cell types, it is more prominent in non-excitable cells, perhaps due, in part, to the inhibition of VGCCs by the essential SOCE proteins STIM1 and STIM2^51,52, raising the possibility that elevated STIM-plasma membrane interactions could have inhibitory effects on mid-capillary pericyte VGCCs. Another interesting possibility is differential inhibition by endogenous polyamines, such as spermine, which has been previously suggested to underlie the decreased functional expression of VGCCs in distal capillary pericytes of the retina³⁰. How these signaling differences translate into functional differences remains another key point of future study.

Although our data suggests Ca²⁺ influx in mid-capillary pericytes occurs primarily via non-voltage-gated channels, this does not exclude a role for pericyte hyperpolarization in functional hyperemia. Several reports on pericyte membrane potential have been made in various preparations from different regions of the CNS, reporting mean resting membrane potentials of between −35 mV and −50 mV^{10,28,37,38,39,50}. In pressurized intact retina preparations, pericytes across the vascular arbor are found more depolarized than smooth muscle cells at low pressure (−43 mV vs. −64 mV)⁵⁰, suggesting pericytes have lower K⁺ permeability at rest. Opening of K_ATP channels increases K⁺ permeability of mid-capillary pericytes, and therefore causes a robust hyperpolarization^37,38,39, which can then be propagated to upstream mural and endothelial cells^37,39. Retrograde hyperpolarization is a robust phenomenon in the microvasculature which rapidly closes VGCCs on upstream SMCs and ensheathing pericytes, thereby decreasing intracellular Ca²⁺ and dilating these contractile cells to increase local cerebral blood flow^{9,27,53,54,55}. Interestingly, we found that blocking K_ATP channels with glibenclamide led to a decrease in the frequency of Ca²⁺ transients, consistent with a decreased driving force for Orai mediated Ca²⁺ influx. This result suggests that K_ATP channels may be open at rest in pericytes from our brain slices, potentially due to increased adenosine tone which can open K_ATP channels via A_2A receptors^37,38. Although modest increases in [K⁺]_ex to 10 mM were previously suggested to decrease Ca²⁺ transient frequency via K_ATP dependent hyperpolarization¹⁸, unlike K_ir2.x channels, K_ATP channels are not known to be activated by [K⁺]_ex. If K_ATP channels were indeed open at rest, as appears to be the case in our study, even modest increases in K⁺ would depolarize pericytes via these open K⁺ channels. Therefore, an alternative interpretation of these results could be that blocking K_ATP channels in their experiments blocked K⁺ induced depolarization rather than hyperpolarization, and thereby reduced driving force for Ca²⁺ entry via non-voltage-gated channels, consistent with our findings. Recordings of mid-capillary pericyte membrane potential changes in response to modest changes in K⁺ concentration are therefore needed to properly interpret these differences.

During neurovascular coupling, ensheathing pericytes of the capillary-arteriole transition zone (which robustly express α-SMA) dilate rapidly, whereas the pericytes of the mid-capillary bed (low or negative for α-SMA) increase their diameter more slowly^9,56. Whereas ensheathing type pericytes dilate actively and independently from the arteriole¹⁰, it remains to be determined whether the mid-capillary diameter increase is a purely passive process or has an active component mediated by mid-capillary pericytes. Interestingly, under pathological conditions such as Alzheimer’s disease and stroke, even mid-capillary pericytes are found to be constricted^28,57, and prolonged optogenetic activation of ChR2 on cortical mid-capillary pericytes has been shown to locally constrict the capillaries that they contact on a slow timescale^8,58. Likewise, increases in intraluminal pressure lead to mid-capillary pericyte depolarization in the retina, and are followed by a delayed constriction⁵⁰. Although these studies suggest that mid-capillary pericyte membrane depolarization is correlated with increased rigidity and tone of the capillary bed, a causal relationship is lacking. Given that our data suggests that mid-capillary pericyte Ca²⁺ influx is dominated by voltage-independent channels, depolarization would be expected to reduce this Ca²⁺ influx and subsequent store filling, due to a decrease in driving force, as in other non-excitable cells. This is consistent with a recent report showing that hyperpolarization of mid-capillary pericytes by application of pinacidil increased pressure-induced Ca²⁺ influx⁵⁰.

Finally, we show here that SOCE is required to amplify the mid-capillary pericyte Ca²⁺ elevation mediated by the vasoconstrictor ET-1. The magnitude of the ET-1-induced Ca²⁺ responses when applied in normal ACSF were highly variable, which could reflect heterogeneous expression of the endothelin-A receptor and/or SOCE proteins in mid-capillary pericytes. ET-1 is a central molecule to pericyte pathology, which is released in ischemia⁵⁹, and which is elevated downstream of Amyloid-β oligomers to constrict pericytes in Alzheimer’s disease⁵⁷. In ensheathing pericytes (1–3rd branch order), the ET-1 evoked [Ca²⁺]_i increase was also recently shown to require an amplification step, but via Ca²⁺ activated Cl^- channel mediated depolarization and L-type VGCC activation²⁸. Our results indicate a similar amplification process in mid-capillary pericytes, but with a clear molecular divergence in the mechanisms mediating ET-1 evoked Ca²⁺ influx. As Ca²⁺ is a ubiquitous signal transduction molecule throughout biology, mediating an array of cellular functions, including gene transcription and contraction, SOCE channels may therefore play an important role in numerous pericyte functions and contribute to their dysfunction in disease.

Study limitations

Genetic tools will ultimately be required to uncover the precise molecular identities underlying mid-capillary pericyte Ca²⁺ signaling. Our pharmacological modulation of SOCE with non-selective (SKF-96365 and 2-APB) and relatively selective (GSK-7975A and IA65) compounds is consistent with the highly Ca²⁺ selective family of Orai channels (namely, Orai1 and Orai3) as the molecular identities underlying pericyte SOCE. However, TRPC channels have been shown to interact with and contribute to SOCE in certain conditions^60,61, although this concept is heavily debated⁶². While our results show that TRPC3/6 are not major contributors, we cannot completely rule out participation of the TRPC1/4/5 subfamily to pericyte SOCE.

The molecular identities of the IP₃R and RyR channels in mid-capillary pericytes will similarly require genetic tools to elucidate. While the IP₃Rs are broadly dispersed throughout cell types, the distribution of RyR isoforms is more segregated, with RyR2 constituting the dominant variant in smooth muscle cells⁶³, making it the most likely variant to be functional in pericytes. Interestingly, RyRs have been classified as functionally absent in most⁶⁴ (but not all^65,66) pericytes of different tissues, including ensheathing type pericytes in the retinal CNS vasculature²⁷. It remains to be determined whether the functional expression of RyRs in cortical CNS mid-capillary pericytes represents a difference across brain regions and/or between pericyte subtypes.

Our study was conducted solely in the brain slice preparation, which is a highly useful tool for probing cellular signaling mechanisms, as pharmacological compounds can be delivered at a controlled concentration and cellular architecture is kept largely intact. However, there are important limitations when compared to in vivo settings: there is no blood pressure and myogenic tone, arterioles and capillaries are collapsed, oxygen concentration (95%) is supraphysiological, and the slice surface has undergone mechanical damage. Translation of this work to in vivo experimentation is an important future direction.

Methods

Ethics statement and animals

All procedures conformed to the guidelines of the Canadian Council on Animal Care and were approved by the “Comité de déontologie sur l’expérimentation animale” (CDEA) of the Université de Montréal (QC, Canada). PDGFRβ-Cre mice⁶⁷ were crossed with Ai95(RCL-GCaMP6f)-D reporter mice (Jackson Laboratory) to obtain PDGFRβ-Cre; GCaMP6f-floxed double transgenic mice. Male and female mice aged P28-P163 were used in experiments.

Acute brain slice preparation

Prior to slicing, mice were put into deep anesthesia with isoflurane and 50 µL of Rhodamine B isothiocyanate-Dextran (70 kDa, 2.5% wt:vol, Sigma-Aldrich) was injected retro-orbitally to label the vessel lumen. Mice were euthanized by decapitation. Following extraction from the skull, brains were placed into ice-cold NMDG-based slicing solution containing (in mM): 120 N-Methyl-d-glucamine, 3 KCl, 25 NaHCO₃, 7 MgCl₂-6H₂O, 1 NaH₂PO₄-H₂O, 20 Glucose, 2.4 Na-pyruvate, 1.3 Na-ascorbate, 1 CaCl₂-H₂O. Then, 300 µm thick coronal cortical slices were cut with a Leica VT 1200S Vibratome. Slices were transferred to a custom chamber with artificial cerebral spinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO₃, 1.5 MgCl₂-6H₂O, 1.3 NaH₂PO₄-H₂O, 10 Glucose, and 1.2 CaCl₂ at 36 °C for 10 min. Slices were then recovered in the chamber at room temperature until use. The fluorescent dye TO-PRO-3 has previously been shown to robustly label mid-capillary pericytes in fixed tissue³⁴, and we have adapted this for imaging in acutely prepared live brain slices. Prior to use, slices were incubated in 1 µM TO-PRO-3 diluted in ACSF for 20 min at room temperature to label and identify the pericyte soma. All solutions used were continuously gassed with 95% O₂ and 5% CO₂.

Pharmacology and ion substitution

All salts were obtained from Sigma-Aldrich. Please refer to Table S3 for identifiers, and vehicles of reagents used. Time 0 represents the start of the first acquisition when the new solution is estimated to have reached the bath (estimated based on the flow rate). For Ca²⁺-free ACSF, CaCl₂ was omitted from the ACSF and 2 mM EGTA was added. For 60 mM K⁺ ACSF, equimolar NaCl was replaced with KCl. Slices were perfused with TTX for 10–20 min prior to imaging. For 60 mM K⁺ experiments, slices were perfused with 60 mM K⁺ for 10-20 min prior to application of VGCC blockers.

Confocal imaging

Imaging was performed with a Zeiss LSM 510 laser scanning confocal microscope with a 40X water immersion objective lens (0.8NA). Pericytes in cortical brain slices were located by TO-PRO-3 and GCaMP6f co-localization and association with a Rhodamine B labeled vessel. GCaMP6f was excited with a 488 nm LED laser and was detected after passing through a 505–530 nm bandpass filter. TO-PRO-3 and Rhodamine B were excited with a 633 nm and 543 nm HeNe laser respectively, which were generally turned off during Ca²⁺ imaging. Ensheathing pericytes in a subset of explicitly labeled experiments were identified based on GCaMP fluorescence almost fully enwrapping the vessel and were definitively confirmed to be within 3 branch orders of the penetrating arteriole. Mid-capillary pericytes were selected for all other experiments on the basis of: (1) Morphology: thin-stand or mesh morphology in which processes clearly did not fully enwrap the vessel (notably different than those found on first 3 branches), (2) being positive for TO-PRO-3 labeling, and 3) the lack of an arteriole detected within 4 branches of the capillary using the Rhodamine B signal. In several cases for mid-capillary pericytes they could not be traced back 4 branches and an arteriole was not visible in the field of view, in these cases only criteria 1 and 2 were used. To measure calcium transient frequency over time, 50 s image acquisitions were made every 3–4 min in frame scanning mode at a 2 Hz sample rate. To record SOCE experiments and [Ca²⁺]_i rises evoked by agonists, a single acquisition was made in frame scanning mode at a 2 Hz sample rate. Slices were perfused with ACSF at 2 mL/min and were kept at 34 ± 2 °C during experiments.

Data collection and analysis

Between frame XY-plane translational movement and within frame distortion were removed using a custom non-rigid alignment MATLAB algorithm, based on the imregdemons (AccumulatedFieldSmoothing = 2.5; PyramidLevels = 4; 32, 16, 8, and 4 iterations respectively for each pyramid level) and imwarp MATLAB functions (https://scanbox.org/2016/06/30/non-rigid-image-alignment-in-twenty-lines-of-matlab/). Hand-drawn regions of interest (ROI) separated pericyte somas from processes. The Astrocyte Quantification and Analysis (AQuA) MATLAB tool³⁵ was used for unbiased event-based Ca²⁺ transient analysis. A Gaussian filter was applied to the images (σ = 2), minimum event size was set to (pixels): 5/pixel size (µm), and detected events occurring in the same frame separated by (in pixels) 1/pixel size (µm) were merged. Event threshold parameters were set to reliably detect the majority of pericyte Ca²⁺ signaling events with minimal detection of false events outside the cell boundaries. Identical analysis parameters were used for all time series of a given cell and experiment. In experiments in which movies were collected at differing timepoints and average data was shown, a linear interpolation between data points was performed. Ca²⁺ transients following agonist application (ET1 or U46619) in Ca²⁺ free solution (Fig. 1f, g) or GSK-7975A (Fig. 6c, d) were only momentarily increased after agonist application before stores became depleted and were therefore analyzed over a brief period during which the agonists visibly evoked an increase in transient signals: ET-1 in Ca²⁺-free (65.2 ± 16.31 s); U46619 (78.86 ± 12.53 s); ET-1 in GSK-7975A (149.1 ± 7.841 s). To compare ET-1 responses at 5, 10, and 30 min in Ca²⁺-free solution (Fig. S2), Ca²⁺ transient frequency following ET-1 application was analyzed over a 50 s timeframe from when the agonist first evoked signals. To examine changes in ensheathing pericyte Ca²⁺ transient frequency following application of 60 mM [K⁺]_ex, an arteriole was located and single 50 s acquisitions of pericytes 1–3 branch orders downstream of the arteriole were taken before and 9–18 min after 60 mM [K⁺]_ex application. Ca²⁺ transient data during all other treatments in Figs. 2–4 was measured from 10–20 min after drug application unless otherwise stated. Transient frequency was normalized to 10 µm of pericyte processes (referred to as ‘length’). To measure SOCE and [Ca²⁺]_i rises evoked by ET-1 in Figs. 5–6, background fluorescence was subtracted, a hand-drawn ROI was placed around the soma, and ΔF/F was averaged 30 s around the peak value recorded. Event density heat maps in figures represent the fraction of frames in which an active event was present in each pixel. To measure resting Ca²⁺ levels (Fig. S4 and Table S1 and S2), a hand-drawn ROI was placed around the soma and an average fluorescence value was calculated from pixels in which AQuA did not detect an event for a given frame. Only cells in which the soma was in focus were included for this analysis. Five cells were excluded due to improper alignment of translational movement between recordings, and six cells were excluded as outliers following identification with the ROUT test (Q = 10%).

Statistics

For all paired data with two groups a two-tailed paired Students’ t test was conducted. For paired data with three groups, a one-way repeated measures ANOVA was conducted, which if significant, was followed by Tukey’s or Šidák’s post-hoc test (see Figure Legends). For all other statistical comparisons normality was first assessed with a Shapiro–Wilk normality test. If this test failed, statistics were calculated by a Kruskal–Wallis non-parametric test, followed by Dunn’s multiple comparisons test.

Software availability

Software used in the study is open source.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data is available with the manuscript as Supplementary Data 1.

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Acknowledgements

This work was supported by an NSERC discovery grant (RGPIN-2020-05276) to R.L.R.; R.L.R. holds a Canada Research Chair in Neurovascular Interactions. B.P. was supported by an USRA award from NSERC and a CIHR MSc Canada Graduate Scholarship. J.C. was supported by an USRA award from NSERC. The authors would like to thank Dr. Richard Robitaille (Université de Montréal) for gifting equipment, Dr. Terry Snutch (University of British Columbia) for gifting Z944, Dr. Mohamed Trebak (University of Pittsburgh) for gifting IA65, Dr. Volkhard Linder (Maine Medical Center) for PDGFRβ-cre mice, and Dr. Isabel Laplante and Pierrette Kwemo for colony and lab management.

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Department of Neuroscience, Université de Montréal, Montréal, QC, Canada
Braxton Phillips & Jenna Clark
Department of Stomatology, Faculty of Dental Medicine, Université de Montréal, Montréal, QC, H3C3J7, Canada
Braxton Phillips, Jenna Clark, Éric Martineau & Ravi L. Rungta
Centre interdisciplinaire de recherche sur le cerveau et l’apprentissage, Université de Montréal, Montréal, QC, Canada
Braxton Phillips, Jenna Clark, Éric Martineau & Ravi L. Rungta

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B.P., E.M., and R.L.R. designed the study. B.P. and J.C. performed experiments. B.P., J.C., and R.L.R. analyzed data and interpreted results. E.M. developed analysis procedures. B.P. and R.L.R. wrote the manuscript with feedback from E.M.; R.L.R. supervised the research. All authors agreed on the final version of the manuscript.

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Correspondence to Ravi L. Rungta.

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Phillips, B., Clark, J., Martineau, É. et al. Orai, RyR, and IP₃R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes. Commun Biol 6, 493 (2023). https://doi.org/10.1038/s42003-023-04858-3

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Received: 30 September 2022
Accepted: 21 April 2023
Published: 06 May 2023
DOI: https://doi.org/10.1038/s42003-023-04858-3

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