Astrocytes drive cortical vasodilatory signaling by activating endothelial NMDA receptors

Chemicals and animals

All chemicals were purchased from Sigma-Aldrich, unless otherwise noted. Procedures involving live animals were approved by the University of Manitoba Animal Care Committee in compliance with the policies of the Canadian Council on Animal Care. All experiments are reported in compliance with ARRIVE guidelines for how to report animal experiments. Conditional silencing of endothelial GluN1 was approached by breeding mice containing dual grin1 alleles flanked by loxP excision sites (grin1^fl/fl; JAX 005246), with mice expressing Cre-recombinase driven by the endothelial promoter Tie-2 (Tek-cre; JAX 008863). Grin1^fl/fl mice expressing Cre-recombinase (grin1^fl/fl · Cre^+/−) have reduced endothelial GluN1 expression, while littermate grin1^fl/fl mice lacking Cre-recombinase (grin1^fl/fl · Cre^−/−) were used as controls. Animals were housed in pairs with a 12-h light/dark cycle and ad libitum access to water and standard chow.

Brain endothelial cell cultures

Brains from neonatal (14–21 days) male and female mice were digested with 0.05% collagenase, re-suspended in 17% dextran and centrifuged at 10,000 × g (30 min, 4℃). Isolated cells were collected on glass beads and cultured on Type I collagen-coated culture dishes in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 2 mM L-glutamine, 1% penicillin-streptomycin, 40 µg/ml heparin and 150 µg/ml endothelial cell growth supplement. Cells were supplemented with 4 µg/ml puromycin for the first 2.5 days, and passaged twice before reaching confluence.

Western blot

Cells or tissues were homogenized in RIPA lysis buffer (50 mM Tris-base, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.25% Na-deoxycholate with protease inhibitors; pH 7.4) and protein concentrations determined using a bicinchoninic acid assay (GE HealthCare). Proteins were separated using sodium dodecylsulfate (SDS)-polyacrylamide (7.5%) gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were exposed to a blocking solution of 5% non-fat powdered milk in TBS with 0.1% Tween-20. Primary antibodies were applied for 12 h (4℃) and included goat anti-GluN1 (1:300, Santa Cruz Biotechnologies), rabbit anti-GluN1 (1:300, Millipore) and goat anti-GluN2C (1:300; Santa Cruz Biotechnologies). Donkey anti-goat or anti-rabbit IgG secondary antibodies, conjugated to horseradish peroxidase (1:1000, Cell Signaling) were applied for 1 h (room temperature), and immunoreactive products visualized using the ECL Plus chemiluminescence kit (GE Life Sciences). Densitometry was performed using ImageJ software (NIH). Values were normalized to protein loading markers.

Immunoprecipitation

Cultured cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer at 4℃ (30 min). Lysates (0.5–1.0 mg) were incubated with 2 µg anti-GluN1 or anti-GluN2C antibodies (Santa Cruz Biotechnologies) at 4℃ (12 h). Protein A/G agarose beads (Thermo Scientific) were added and mixed gently at 4℃ (4 hours). Beads were then precipitated (2500 × g for 3 min) and washed with RIPA buffer. Complexes were then eluted with 50 µl RIPA buffer containing 2.5% SDS and boiled at 95℃ (30 min). Proteins were resolved using SDS-polyacrylamide gel electrophoresis and analyzed by Western blot.

Immunogold electron microscopy

Immunoelectron microscopy was performed as described previously. ³⁶ Neonatal (14–21 day old) mice were anesthetized with ketamine (150 mg/kg) and xylazine (15 mg/kg), and perfused through the left ventricle with PBS, followed by 4% paraformaldehyde (PFA). Cortical volumes (1 mm³) were cryoprotected in sequentially higher glycerol concentrations (10–30% wt./vol), and plunge-frozen in liquid ethane (−180℃). Tissue was fixed en bloc with 1.5% uranyl acetate in methanol (−90℃, 30 h), and methanol progressively replaced by HM-20 embedding resin (Electron Microscopy Sciences) over 48 h (−45℃). Resin was then UV-polymerized for 48 h, and ultrathin sections were cut and collected on formvar-coated grids. Grids were etched with a solution of 0.1% sodium borohydride, followed by 50 mM glycine, washed in TBST (50 mM Tris-base, 150 mM NaCl, 0.1% Triton-X; pH 7.4), and blocked with 2% (wt./vol) bovine serum albumin in TBST. Grids were incubated overnight with primary antibodies (rabbit anti-GluN1, Cell Signaling; mouse anti-PSD-95, BD Biosciences), washed with TBST and incubated for 2 h with gold bead-conjugated secondary antibodies (anti-mouse, 15 nm; anti-rabbit 10 nm; Aurion). Grids were lightly counterstained with 2% (wt./vol) uranyl acetate and Reynold’s lead. GluN1-labeled gold particles within 30 nm of either cell membrane were considered to be associated with that structure.

Measurement of intracellular NO production

Endothelial cells were treated with glutamate and D-serine for 5 h, washed with modified Hanks-Buffered Saline Solution (HBSS; 140 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM Glucose, 20 mM HEPES) and loaded with the NO fluorescent indicator, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM, 4 µM; Invitrogen) for 50 min (37℃). DAF signal was assessed using a Zeiss laser scanning microscope with 20× objective and an argon laser delivering an excitation wavelength of 495 nm. Emitted signal (515 nm) was captured and analyzed using ImageJ. NO production was expressed as relative fluorescence (F/F₀) of DAF-FM, where F is fluorescence intensity of treatment groups and F₀ is baseline fluorescence intensity of untreated cells.

Quantitative PCR

Total RNA was isolated using the TRIzol Reagent (ThermoFisher Scientific), and analyzed using the iScript One-Step RT-PCR Kit with SYBR Green dye (Bio-Rad) and an Applied Biosystems 7300 RT-PCR system. β-actin (internal control) and GluN1 were amplified using the following primers: β-actin forward, 5′-GGGCTATGCTCTCCCTCACG-3′; β-actin reverse, 5′-GTCACGCACGATTTCCCTCTC-3′; GluN1 forward, 5′-GTGCCAAACTTGCCATCTGC-3′; GluN1 reverse, 5′-CGGGGCCTAATGACACATCC-3′. PCR parameters were as follows: 50℃ for 10 min, 95℃ for 1 min, followed by 50 cycles at 95℃ for 15 s and 60℃ for 60 s. Relative amount of target gene mRNA was normalized to β-actin mRNA. Standard curves were generated and the relative amount of target gene mRNA was normalized to β-actin mRNA. Specificity was verified by melt curve analysis.

Brain slices and two-photon laser scanning microscopy

Brains from day 14–19 postnatal male and female mice were placed in ice-cold cutting buffer (2.5 mM KCl, 1.25 mM NaH₂PO₄, 10 mM MgSO₄, 5 mM CaCl₂, 26 mM NaHCO₃, 10 mM glucose, 230 mM sucrose) with 95% O₂ and 5% CO₂. Slices (350 µm) were cut using a vibrating blade, and maintained in artificial cerebrospinal fluid (aCSF: 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, 10 mM glucose) equilibrated with 95% O₂ (35℃). Slices were treated with the Ca²⁺ indicator dye, rhodamine-2 AM (rhod-2, 10 µM; Invitrogen), with or without the caged Ca²⁺ compound, o-nitrophenyl-EGTA AM (NP-EGTA, 10 µM; Invitrogen) and/or Griffonia simplicifolia 1 Isolectin B₄ tagged with Alexa Fluor 488 (5 µg/ml; Invitrogen) for 1 h before imaging (37℃). In some experiments, DAF-FM diacetate (25 µM, Invitrogen) was bath-applied in aCSF for 30 min prior to rhod-2 exposure in addition to the rhod-2 incubation period. For imaging, slices were superfused in open chambers with aCSF equilibrated with 20% O₂, 5% CO₂, and 75% N₂. Slices were imaged using a two-photon laser scanning microscope (TPLSM) with excitation wavelength of 800 nm delivered by a Ti-sapphire laser (Coherent) through an Ultima multiphoton scan head with dual galvanometers for imaging and uncaging (Bruker). Emitted signal was collected by photomultiplier tubes at 488 nm (DAF-FM and isolectin) or 580 nm (rhod-2).

In selected experiments, glutamate and D-serine (1 mM each, 0.2 µl) were delivered by 40 ms picospritzer pressure ejection (10 kPa) from glass micropipettes to discrete areas of cortical penetrating arterioles. Micropipette tips with an internal tip diameter of 1–10 µm were positioned 10 µm from arteriolar walls. For astrocytic Ca²⁺ uncaging, functional mapping software (TriggerSync, Bruker) was used to guide a 700 nm/200 ms laser pulse through a voltage-controlled pockels cell (laser power 15–20 mW). Images were analyzed every 15 s using Prairie View software to determine relative changes in lumen diameter, marked by isolectin staining and DODT laser contrast images. All vasodilatory responses were assessed with no agent provided to enhance baseline smooth muscle tone. Responses were similar to those observed in slices pre-treated with 2 µM norepinephrine (Supplemental Material).

PGE₂ and 20-HETE quantification

Acute brain slices were incubated in aCSF with 20% O₂/5% CO₂ and exposed to tACPD (100 µM, Tocris Bioscience) or glutamate with D-serine (10 µM each) for 5 min. ¹¹ PGE₂ release over 5 min was measured using a PGE₂ enzyme immunoassay Kit (Cayman). Tissue 20-HETE content was quantified using a 20-HETE ELISA Kit (Detroit R&D).

Statistics

All data are presented as means ± standard error of the mean (SEM). Statistical analysis was performed throughout using GraphPad Prism version 5.0. Unpaired, two-tailed t-tests were used to compare two groups with a single variable. One-way ANOVA with Newman–Keuls post hoc test was used to compare multiple groups. Area under curve (AUC) was calculated from 5-min time course assessments for vessel diameter changes, rhod-2 signal and DAF-FM signal. AUCs were compared using t-tests for two groups. Projected sample sizes were calculated assuming a standard deviation of 15% and a minimum detectable mean difference of 30%. Acceptable alpha error was set at 0.05 and the acceptable beta error at 0.80. Data collectors were blinded to genotype and treatment groups in all immuno-EM experiments, where manual counting was required.

Brain endothelial cells express NMDA receptors linked to eNOS activity

Immunoreactivity for the pan-NMDA receptor subunit, GluN1, was detected in primary brain microvascular endothelial cells. Anti-GluN1 (Santa Cruz) was used to immunoprecipitate (IP) total homogenates from endothelial cultures and cerebral cortex, and immunoblotted products were detected with a separate anti-GluN1 (EMD Millipore, Figure 1(a)). IP products were also immunoreactive for GluN2C (Figure 1(a)), indicating a physical linkage between GluN1 and GluN2C. These results were confirmed using reciprocal IP experiments in which GluN2C IP products were immunoreactive for GluN1. To determine whether expression of NMDA receptor subunits correlates with functional NMDA receptors, endothelial cultures were loaded with the NO-sensitive dye, DAF-FM, and exposed to acetylcholine or NMDA receptor co-agonists, glutamate and D-serine (Figure 1(b), green). Glutamate enhanced DAF-FM fluorescence in the presence of 10 µM D-serine but not alone (Figure 1(c)). Glutamate concentrations as low as 3 µM significantly elevated DAF-FM signal (143 ± 11% baseline), with a maximum effect of 264 ± 21% at 300 µM. The NOS inhibitor, L-NIO, virtually eliminated DAF-FM signal induced by glutamate (30 µM) and D-serine (10 µM), as well as by acetylcholine (Figure 1(d)). The glutamate/D-serine effect on NO generation was also significantly reduced by NMDA receptor glutamate site antagonist, D-2-amino-5-phosphonopentanoate (AP5), and glycine site antagonist, 5,7-dichlorokynurenic acid (DCKA, Figure 1(e)). In contrast, neither AP5 nor DCKA affected NO generation produced by acetylcholine. NO production in response to glutamate/D-serine and acetylcholine was also mitigated by limiting accumulation of intracellular Ca²⁺ (Figure 1(f)). Loading cultures with the Ca²⁺-chelator, BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis]-AM, reduced NO accumulation from 184 ± 10% to 131 ± 10% control, and in response to acetylcholine from 234 ± 14% to 107 ± 5% control. Similarly, agonist treatment in Ca²⁺-free media with 0.1 mM EGTA reduced the effect of glutamate/D-serine to 101 ± 5% control and the effect of acetylcholine to 93 ± 12% control. Together these data show that primary brain endothelial cultures express NMDA receptors capable of activating NOS in a Ca²⁺-dependent fashion. OPEN IN VIEWER

GluN1 is localized to basolateral brain endothelial membrane surfaces

We used immunoelectron microscopy to examine the subcellular endothelial GluN1 distribution in cortical slices. Anti-GluN1 was conjugated to gold beads with 10 nm diameter to create a detection system that indicates the presence of target epitope within a 30 nm radius (Figure 2(a)). ³⁶ Cortical synapses were first examined to confirm GluN1 detection. Tissues were exposed to both anti-GluN1 (10 nm bead) and an antibody against PSD-95 (15 nm bead), which is a synaptic protein that associates with the GluN1 C-terminus. ³⁷ GluN1-labeled cortical synapses were dominantly asymmetric (97.7%) and exhibited strong association with PSD-95 (Figure 2(b)). The vast majority of GluN1 was localized to within 30 nm of postsynaptic membranes (Figure 2(c)), verifying that anti-GluN1 faithfully labels NMDA receptors. We next quantified GluN1 beads within 30 nm of basolateral endothelial membranes. GluN1 was detected at basolateral and apical endothelial membranes (Figure 2(d) to (g)). For cortical arteries, arterioles and capillaries, apical GluN1 expression was significantly less, proportionally, relative to basolateral expression. Basolateral expression represented 73 ± 15%, 77 ± 4% and 71 ± 4% of GluN1 immunoreactivity in arteries, arterioles and capillaries, respectively (Figure 2(h)). These data demonstrate that NMDA receptor subunits are expressed with membrane polarity suitable for direct communication with the brain compartment. OPEN IN VIEWER

Astrocytic activation leads to cortical vasodilation mediated by endothelial NMDA receptors

In neonatal cortical slices, hyperemic signaling can be simulated by bath-applying trans-1-aminocyclo-pentane-1,3-dicarboxylic acid (tACPD), which activates astrocytic mGluR5 receptors and leads to astrocyte-mediated local vasodilation.^8,11 Cortical layer II/III arteriolar segments with astrocytic endfeet were chosen for analysis by two-photon laser scanning microscopy (TPLSM) in acute cortical slices (Figure 3(a)). Astrocytic Ca²⁺ (Figure 3(b); rhodamine-2, green) and segmental arteriole lumen diameter were assessed every 15 s prior to and after tACPD application. Lumen diameter increased steadily to a peak of 5.2 ± 0.7% over baseline diameter at 105 s, followed by a return to baseline by 5 min (Figure 3(c)). In a cumulative analysis of this 5 min interval, assessed as area under the curve (AUC), the competitive glutamate site NMDA receptor antagonist, AP5 (50 µM), reduced the vasodilatory response by 74% (from 825 ± 135 to 211 ± 81, Figure 3(d)). Similarly, the co-agonist site NMDA receptor antagonist, DCKA (100 µM), attenuated tACPD-induced vasodilation by 67% (from 1063 ± 186 to 349 ± 121). OPEN IN VIEWER

To more directly understand the role of endothelial NMDA receptors in neurovascular coupling, we developed a selective endothelial loss of function model by crossing floxed grin1 (gene for GluN1, grin1^fl/fl) mice with mice expressing Cre-recombinase driven by the Tie-2 promoter (Tie-2-Cre^+/−). Grin1^fl/fl · Cre^+/− offspring (Cre⁺) are animals with conditional GluN1 silencing, while littermate grin1^fl/fl · Cre^−/− offspring (Cre⁻) are controls. In Cre⁺ brain endothelial cultures, mRNA for GluN1 was reduced to 27 ± 10% control levels (Figure 4(a)), and GluN1 immunoreactivity was reduced to 49 ± 10% of control expression (Figure 4(b)). In agreement, NO generation in cultures treated with glutamate and D-serine was reduced from 209 ± 65% control in Cre⁻cultures, to 96 ± 6% control in Cre⁺ cultures (Figure 4(c)). By contrast, GluN1 loss of function did not affect NO generation induced by acetylcholine. Using quantitative PCR in cortical homogenates, we found no difference in GluN1 transcript levels between Cre⁺ and Cre⁻ mice, likely reflecting a proportional dominance of neuronal GluN1 expression in brain tissue (Figure 4(d)). We also used mesenteric artery as a peripheral vascular tissue free of neuronal GluN1 and found that Tie-2-Cre expression reduced GluN1 transcript to 7.5 ± 0.5% of control Cre⁻ tissues (Figure 4(d)). Synaptic and endothelial GluN1 immunoreactivities were examined using immunoelectron microscopy. GluN1-positive gold bead density in the basolateral endothelium declined from 1.24 ± 0.06 beads/µm of membrane surface area in control cortex, to 0.543 ± 0.001 in Cre⁺ cortex (Figure 4(e)), whereas synaptic GluN1 immunoreactivity did not change. These results indicate selective, partial loss (>50%, protein level) of GluN1 in endothelial cells. OPEN IN VIEWER

In Cre⁻ control brain slices exposed to bath-applied tACPD, arteriolar lumen diameter increased to a peak of 5.0 ± 1.3% over baseline at 225 s, followed by a return to baseline by 5 min (Figure 5(a)). Cumulatively, vasodilatory responses (AUC) were reduced by 54 ± 9% in Cre⁺ slices with endothelial NMDA receptor loss of function (Figure 5(b)). By contrast, astrocytic Ca²⁺ levels increased rapidly in both Cre⁺ and Cre⁻ slices in response to tACPD (Figure 5(c)), with no significant difference in cumulative responses between groups (Figure 5(d)). This indicates differences in vasodilatory effects result from events downstream of astrocytic activation. We also directly applied NMDA receptor co-agonists, glutamate and D-serine together, by pressure ejection 10 nm adjacent to cortical arterioles (1 mM each agonist). While this treatment initiated a peak vasodilatory response of 3.6 ± 0.8% of baseline in Cre⁺ slices, the peak effect was smaller and return to baseline more rapid than in Cre⁻ wildtype controls (Figure 5(e)). This resulted in a significantly smaller cumulative vasodilatory effect in endothelial Cre⁺ slices (Figure 5(f)). OPEN IN VIEWER

To more directly study linkage between astrocytic activity and endothelial NMDA receptors, acute cortical slices were loaded with the caged Ca²⁺ compound, NP-EGTA, and perivascular astrocytes targeted for two-photon flash photolysis (TPP). Similar to tACPD and NMDA receptor agonists, selective activation of single astrocytes increased lumen diameter in Cre⁻ wildtype slices (Figure 5(g), Supplemental video 1). The cumulative vasodilatory response was reduced by 70 ± 18% with endothelial NMDA receptor silencing (Figure 5(h)). Overall these data demonstrate that mouse cortical slice arterioles with partial endothelial NMDA receptor silencing have diminished ability to dilate in response to multiple stimulation paradigms.

Direct activation of cortical astrocytes leads to endothelial NO generation dependent on endothelial NMDA receptors

To determine whether astrocytes communicate with the endothelium to produce NO, cortical slices were loaded with DAF-FM, NP-EGTA-AM and rhodamine-2-AM. Layer II/III cortical arterioles with adjacent perivascular astrocytes were then imaged by TPLSM. Single, rhodamine-2-labelled astrocytes were targeted for activation by TPP (Figure 6(a), white asterisks). TPP rapidly enhanced astrocytic rhodamine-2 fluorescence (Figure 6(a), green), reflecting increased intracellular Ca²⁺ levels. In control slices, TPP also enhanced DAF-FM signal intensity, indicating NO accumulation (Supplemental Video 2). This signal was observed in the endothelium, closer to the arteriolar lumen than the astrocytic rhod-2 signal. While indicative of NO accumulation, strong DAF-FM signals dampened vasodilatory efficacy and in some cases were associated with vasoconstriction, likely by inactivating NO (e.g. Supplemental Video 2). Arterioles were also identified that dilated in response to TPP but had weaker DAF-FM signals (e.g. Supplemental Video 3), suggesting vasodilatory activity was inversely related to DAF-FM loading in individual vessels. Astrocytic rhod-2 signal increased rapidly, rising to 161 ± 19% of unstimulated values within 3 s after TPP and to 196 ± 17% by 15 s (Figure 6(b)). Endothelial DAF-FM signal increased more slowly, reaching a maximum of 177 ± 33% of unstimulated control values by 225 s after TPP (Figure 6(a) and (b)). TPP was unable to generate an endothelial NO/DAF-FM signal in the presence of the NOS inhibitor, N⁵-(1-iminoethyl)-L-ornithine (L-NIO, 3 µM; Figure 6(c) and (d); also Supplemental video 3). These data confirm direct communication between astrocytes and endothelial cells, resulting in endothelial NO generation. OPEN IN VIEWER

NO levels were assessed in response to astrocytic Ca²⁺ uncaging in slices from Cre⁻ control and endothelial Cre⁺ mice. In Cre⁻ slices loaded with DAF-FM, TPP induced a cumulative DAF-FM signal that was significantly inhibited by competitive NMDA receptor antagonists, AP5 and DCKA, and by Tie-2-Cre expression, but not by the AMPA receptor antagonist, CNQX (Figure 7(a)). Vasodilatory responses induced by TPP of NP-EGTA in astrocytes were significantly inhibited by the guanylyl cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 3 µM) in Cre⁻ control cortical slices but not in Cre⁺ slices with endothelial NMDA receptor loss of function (Figure 7(b)). We also determined whether endothelial NMDA receptors affect the balance of arachidonic acid metabolites known to cause smooth muscle relaxation (PGE₂) and constriction (20-HETE). Cortical slices were exposed to bath-applied tACPD (100 µM), and 20-HETE and PGE₂ quantified by ELISA after 5 min. While 20-HETE levels were unaffected in Cre⁻ controls, there was significant enhancement of 20-HETE in Cre⁺ slices with endothelial NMDA receptor silencing (Figure 7(c)). In contrast, PGE₂ release was significantly elevated following tACPD treatment of Cre⁻ wildtype slices, and this affect was not significant with more than 95% certainty in Cre⁺ slices (Figure 7(d)). There was also a significant decline in induced PGE₂ levels when endothelial GluN1 was silenced, suggesting a source of PGE₂ dependent on endothelial NMDA receptor activity. Vasodilatory responses to TPP of NP-EGTA in astrocytes were significantly reduced by the cyclooxygenase inhibitor, indomethacin (100 µM) in Cre⁻ cortical slices, indicating that endothelial NMDA receptors affect arteriolar lumen diameter by a mechanism dependent on a vasodilatory arachidonic acid metabolite like PGE₂ (Figure 7(e)). While the vasodilatory magnitude was dramatically reduced by endothelial GluN1 silencing, the remaining response was still significantly sensitive to inhibition by indomethacin. OPEN IN VIEWER