Abstract
NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (INMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). INMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (Vholding) −70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (IPEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (Iholding) at Vholding −70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the IPEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger IPEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered INMDA at Vholding +40 mV under 1.2 mM [Mg2+]o condition. Iifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.
Significance Statement
The NMDAR-mediated excitatory tone between cells is transmitted via phasic activation of synaptic NMDARs (EPSCs) and tonic activation of extrasynaptic NMDARs (INMDA) in the brain. The activation of NMDARs depends on the glutamate concentration, NMDAR subunit composition, and their subcellular localization, as well as the membrane potential. Therefore, the mechanism of NMDAR-mediated excitatory tone varies in different pathophysiological conditions. Our results show that the INMDA in nondepolarized and depolarized neurons is dominantly mediated by NR2A- and NR2B-containing NMDARs, respectively, and the former efficiently detects the ambient glutamate concentration in the supraoptic magnocellular neuroendocrine cells of normal and hypertensive rats. This study shows that NR2A-containing NMDARs could be a biosensor detecting ambient glutamate concentration in the brain.
Introduction
Glutamate is a major excitatory neurotransmitter in the supraoptic nucleus (SON; van den Pol et al., 1990), which is composed of vasopressin and oxytocin magnocellular neurosecretory cells (MNCs). These neurons are known to play critical roles in fluid balance and cardiovascular and reproductive homeostasis (Silverman and Zimmerman, 1983). Neurohumoral activation has a direct impact on morbidity/mortality in cardiovascular diseases (Cohn et al., 1984; Yemane et al., 2010). In addition to the classical transient excitatory postsynaptic currents (EPSCs) mediated by synaptic receptors, glutamate generates a tonic, sustained excitatory current (INMDA) when it binds to extrasynaptic NMDARs (eNMDARs) that strongly stimulate firing activity in SON MNCs (Fleming et al., 2011). NMDARs are heterotetramers composed of two NR1 subunits and two NR2 subunits. NMDARs containing NR2A, B, C, and D subunits encoded by four different genes (GluN2A-D) exhibit distinct electrophysiological and pharmacological properties as well as different subsynaptic distributions and expression profiles. For example, eNMDARs containing NR2B or NR2D subunit could mediate INMDA with their tonic activation (Fleming et al., 2011; Neupane et al., 2021), while NR2A-containing NMDARs, predominantly found in synaptic space, mediate EPSCs. NR2D-containing eNMDARs even generate a “Mg2+-resistant” INMDA activated in nondepolarized SON MNCs under the physiological concentration of [Mg2+]o (Neupane et al., 2021). Given that elevated glutamatergic excitatory tone supports exacerbated activity of MNCs (Biancardi et al., 2010; Li et al., 2014; Glass et al., 2015; Zhang et al., 2017), which in turn contributes to neurohumoral activation during cardiovascular diseases, elucidating the precise roles of different NMDARs and their subunit plasticity altering MNCs activity in diseases such as hypertension and heart failure is of critical importance.
Under conditions requiring strong secretion of neurohypophysial hormones, there is a pronounced reduction in the astrocytic coverage of SON NMCs (Theodosis and Poulain, 1993; Bobak and Salm, 1996). Neuroglia remodeling in response to physiological challenges resulted in blunted glutamate transporter (GLT) activity leading to increased ambient glutamate in the SON (Fleming et al., 2011), which may depolarize the neurons despite the Mg2+ block of NMDARs (Mayer et al., 1984; Nowak et al., 1984). Given that robust NR2D protein expression in the SON MNCs is uncommon in the adult brain (Doherty and Sladek, 2011) and that NR2D generates an “Mg2+-resistant” INMDA of MNCs in a state-dependent manner (Neupane et al., 2021), we investigated whether NR2D-containing eNMDARs contribute to exacerbated INMDA in SON neurons during hypertension and whether this finding could be a mechanism contributing in turn to neurohumoral activation in this cardiovascular disease. Unexpectedly, our results demonstrate that in DOCA-salt hypertensive rats, a decreased activity of excitatory amino acid transporters (EAATs) resulted in increased ambient glutamate levels to potentiate a Mg2+-sensitive INMDA mediated by NMDAR-containing NR2A rather than NR2B and NR2D.
Materials and Methods
DOCA-salt hypertension model
All animal experimentation was approved by the Institutional Animal Care and Use Committee and was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male 5-week-old Sprague Dawley (SD) rats weighing 120–180 g were purchased from Animals. All animals were housed on a 12-hour light/dark cycle and had access to food ad libitum throughout the experiments. After 1 week of acclimatization, rats were anesthetized with avertin (250 mg/kg, i.p.; Millipore Sigma) and underwent unilateral nephrectomy (left kidney) as previously described (Prahalathan et al., 2012). Briefly, a lateral abdominal incision was made to access the left kidney for its resection and sutured after the removal of the kidney. After 1 week, DOCA (D7000-5G, Sigma-Aldrich) was implanted subcutaneously in all nephrectomized rats, and animals were randomly assigned to either the H2O group (DOCA-H2O) or salt group (DOCA-salt). The DOCA-salt group had their water replaced with a mixture of 0.8% NaCl and 0.2% KCl in tap water to drink until killed (after 1 or 4 weeks).
Blood pressure measurement
Systolic blood pressure was measured once a week for a month by tail cuff method as previously described (Kubota et al., 2006). Rats were restrained in a cylindrical restrainer at 37°C for 30 min to acclimatize them to the apparatus (noninvasive blood pressure system, CODA; Kent Scientific Corporation) before blood pressure recordings were made. Systolic blood pressure was measured in awake rats using a noninvasive tail cuff blood pressure measuring system (PowerLab/8SP data acquisition system, ADInstruments) before DOCA treatment and on the 1, 2, 3, and 4 weeks of DOCA treatment. The physiological data were analyzed using the LabChart 6.1 Pro software (ADInstruments). Averaged blood pressure from at least five consecutive readings obtained from each rat was recorded as final blood pressure.
Water intake, urine output, serum osmolality, and urine osmolality measurements
After 6th day of DOCA treatment, both DOCA-H20 and DOCA-salt groups were individually housed in metabolic cages provided with H2O or salt for 24 hours before sample collection. Urine output and water intake were measured within the following 24 hours. In addition, on the 7th day, a fresh urine sample was collected in a tube, and animals were anesthetized with avertin (250 mg/kg, i.p.; Millipore Sigma) for blood collection by cardiac puncture. Both urine and blood were centrifuged. After centrifugation, urine and serum osmolality were measured using the freezing point depression method and a micro-osmometer (model 210, Fiske Associates).
Electrophysiology and data analysis
Patch-clamp recordings from SON MNCs were obtained from acutely prepared hypothalamic slices (300 µm) as previously described (Neupane et al., 2021). Briefly, rats were decapitated under avertin anesthesia (Avertin, 200 mg/kg, i.p.), and the brains were quickly extracted. Sectioned slices were incubated in artificial CSF (aCSF) containing (in mM) 126 NaCl, 5 KCl, 1.2 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, 10 glucose, and 2.4 CaCl2, pH 7.3–7.4, and saturated with 95% O2 and 5% CO2 within the slice holder for 1 h at 34°C in the presence of 3 µM glutamic acid. Single hemisectioned slices were transferred to a recording chamber perfused with aCSF saturated with 95% O2 and 5% CO2 and maintained at 34°C. All electrophysiological measurements were recorded using a MultiClamp 700B (Molecular Devices). The current output was filtered at 1 kHz and digitized at 10 kHz (Digidata 1440 and pClamp 10.2 software, Molecular Devices). Data were excluded if the series resistance was not stable throughout the entire recording (20% change) or if neuronal input resistance (IR) was <550 MΩ at the beginning of the recording. The NMDA receptor-mediated tonic current (tonic INMDA) was defined as changes in the holding current (Iholding) in the presence of ionotropic GABA receptor antagonists and was calculated by the difference in Iholding measured as the average of a 2-minute steady-state baseline segment obtained before and after the application of NMDAR antagonists. INMDA was recorded and calculated at −70 mV with low [Mg2+]o (20 µM) or +40 mV with normal aCSF containing 1.2 mM [Mg2+]o unless otherwise stated. Event detection and analysis of spontaneous EPSCs were carried out using MiniAnalysis software (Synaptosoft) at Vholding −70 mV, as previously described (Park et al., 2007). The detection threshold was set at −20 pA and 75 pA/ms for EPSC amplitude and area, respectively. From extracted EPSCs, frequency, amplitude, and decay time constant were calculated. EPSC decay time constants were calculated from single exponential fits.
Drugs were added to the aCSF perfusing solution at differing concentrations. The final concentration of dimethylsulfoxide (DMSO) was <0.05%, when used as a vehicle. DL-2-amino-5-phospho-nopentanoic acid (AP5), (2S*, 3R*) -1-(phenanthren-2-carbonyl) piperazine-2, 3-dicarboxylic acid (PPDA), [[[(1S)-1-(4-bromophenyl)ethyl]amino](1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl] phosphonic acid tetrasodium salt (PEAQX) and DL-threo-β-benzyloxyaspartic acid (TBOA) were all purchased from Tocris Bioscience. All other drugs were purchased from Sigma-Aldrich.
Western blotting
Brain tissue punches containing the SON were collected from 300 µM coronal hypothalamic slices (three sequential slices/rats) from each brain as described previously (Potapenko et al., 2012). Protein was extracted from the SON punches using a mixture of a protease inhibitor and radioimmunoprecipitation assay (RIPA) lysis buffer and quantified using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific). Approximately 50 µg of protein was separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were then blocked with TBST (0.1% Tween 20 in 1× Tris-buffered saline) containing 5% skimmed milk for 1 h at room temperature. The blot was then probed with primary antibodies against NMDAR NR2A (1:1,000; catalog #AGC-002, Alomone Labs; RRID: AB_2756596), NR2B (1:1,000; catalog #06-600, Millipore; RRID: AB_310193), and NR2D subunits (1:1,000; catalog #AGC-020, Alomone Labs; RRID: AB_10658334) overnight at 4°C. Next, the membranes were exposed to horseradish peroxidase-conjugated goat anti-rabbit (catalog #7074, Cell Signaling Technology; RRID: AB_2099233) and anti-mouse (catalog #31430, Thermo Fisher Scientific; RRID: AB_228307) secondary antibody (1:1,000) at room temperature for 1 h. Proteins were visualized using a pierce enhanced chemiluminescence detection kit (Thermo Fisher Scientific), and the intensity of the bands was measured using ImageJ software 1.42q (National Institutes of Health).
Statistical analysis
Numerical data are presented as the mean ± SEM. To assess the differences in tonic INMDA under DOCA-salt conditions, we performed a hierarchical testing procedure. In the first step, a Shapiro–Wilk test was used to test the null hypothesis that the data distribution was normal with a significance level of 5%. For data with a normal distribution, the statistical significance of comparisons was assessed using either a two-sample t test or a one-way ANOVA followed by a post hoc test (e.g., Bonferroni's test). If the null hypothesis was rejected, nonparametric tests were used with Microcal Origin software (RRID: SCR_002815). For all experiments, male rats were used to avoid effects of hormonal changes on the results. Tonic INMDA was categorized into two modalities: either the Mg2+-sensitive tonic INMDA, measured by Iholding shift induced by increasing the extracellular Mg2+ concentration from 20 μM to 1.2 mM at Vh, −70 mV, or the Mg2+-resistant tonic INMDA, measured by Iholding shift induced by NMDAR antagonists at Vh, −70 mV in normal aCSF. Electrophysiological recordings were taken from three or more animals per group, and three to four slices were collected per animal. The Mann–Whitney test was used to compare two groups if the data were not normally distributed. The pharmacological sensitivity of tonic INMDA to various NMDAR antagonists was investigated. To compare INMDA amplitude and protein expression (NR2A, NR2B, and NR2D) in the H2O group and DOCA-salt group, we used one-way ANOVA followed by a post hoc test.
Results
Transient increase of INMDA in SON MNCs in DOCA-salt rats
DOCA-salt treatment successfully induced hypertension as shown by a time-dependent increase in systolic arterial pressure (SAP) in rats. The nephrectomy/DOCA-salt treatment (DOCA-salt) elicited a significant increase in the SAP (F(2,72) = 48.03; p = 0.01; two-way ANOVA). SAP tended to increase at 1 week, increased significantly at 2 weeks, reached the maximal hypertension at 3 weeks, and was maintained up to 4 weeks post-DOCA implantation (PDI) in DOCA-salt group, while the nephrectomy/DOCA alone with no NaCl and KCl (DOCA-H2O) did not affect SAP during the period compared with normal control animals (p = 0.62; Bonferroni’s post hoc test following two-way ANOVA; Fig. 1A).
Increased INMDA of SON MNCs in DOCA-salt rats. A, Systolic blood pressure at different times in naive control (normal control, n = 6), DOCA-H2O (n = 6), and DOCA-salt model rats (DOCA-salt, n = 18) (*p < 0.05, ***p < 0.001, compared with normal control). B, Representative current traces showing effects of Mg2+, followed by the sequential application of AP5 (200 µM), a NMDA receptor antagonist, in holding current in DOCA-H2O and DOCA-salt model rats at 1 week. Representative current traces show the effect of sequential application of Mg2+ (1.2 mM) and additional AP5 (100 mM) on the holding current of SON MNCs at 1, 2, and 4 weeks. C,D, Summarized bar graph showing average tonic current amplitude block by Mg2+ (C) and additional AP5 (D) in each week, respectively. **p < 0.01, compared with DOCA-H2O.
Increased plasma vasopressin (VP), a neurohypophysial hormone, level may affect the fluid homeostasis (Boone and Deen, 2008; Parekh et al., 2021). Although SAP increase did not reach statistical significance at 1 week PDI (DOCA-H2O 119.34 ± 0.975 mmHg, n = 5 vs DOCA-salt 123.99 ± 2.69 mmHg, n = 18 rats), the volume of water intake and urine output were significantly increased in DOCA-salt than DOCA-H2O groups at 1 week PDI (Table 1). In addition, the urine osmolality was significantly lower in DOCA-salt than that in DOCA-H2O rats, meanwhile the serum osmotic pressure was not different in the two groups (Table 1).
Changes in metabolic parameters after 1 week in DOCA-salt induced hypertensive model
In next experiment, we investigated INMDA in SON MNCs in DOCA-H2O and DOCA-salt groups at 1, 2, and 4 weeks PDI. Increasing [Mg2+]o from 20 µM to 1.2 mM induced a significantly larger Iholding in DOCA-salt (15.17 ± 2.32 pA, n = 19 neurons from 7 rats) compared with DOCA-H2O group (6.25 ± 0.98 pA, n = 9 neurons from 3 rats) at 1 week PDI (Fig. 1B,C), while the difference was not observed at 2 and 4 weeks PDI. An additional NMDAR antagonist, AP5, failed to cause further Iholding shift in all tested groups (Fig. 1B,C). These results suggested that more NMDARs could be activated to generate Mg2+-sensitive INMDA in SON MNCs in DOCA-salt rats at 1 week PDI.
Pharmacology of the enhanced INMDA in DOCA-salt rats
To assess the composition of NMDARs that potentiate Mg2+-sensitive INMDA at 1 week PDI, we investigated INMDA at Vholding −70 mV in low Mg2+ aCSF and compared its sensitivity with NR2A, NR2B, and NR2C/D subunit selective antagonists in DOCA-H2O and DOCA-salt group (Fig. 2).
Pharmacology of INMDA in DOCA-H2O and DOCA-salt group. A–C, Representative current traces showing the effect of PEAQX (1 µM), a NR2A receptor antagonist (A), ifenprodil (30 µM), an NR2B receptor antagonist (B), and PPDA (1 µM), an NR2C/D antagonist (C) on holding current. Note that ifenprodil and PPDA caused minimal effects on Iholding, while additional PEAQX caused larger Iholding changes in DOCA-salt groups. D, Summarized bar graph showing mean of holding current change by each antagonist. *p < 0.05, compared with DOCA-H2O; n = 6 and 8 in DOCA-H2O and DOCA-salt, respectively.
PEAQX, an NR2A subunit antagonist, uncovered INMDA (IPEAQX) of nondepolarized SON MNCs under low Mg2+ condition in both DOCA-H2O group (7.84 ± 0.92 pA; n = 6 neurons from 3 rats; F(2,15) = 16.23; p = 0.003; Bonferroni’s post hoc test following one-way RM-ANOVA) and DOCA-salt (14.80 ± 2.49 pA; n = 8 neurons from 3 rats; F(2,15) = 33.14; p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA). IPEAQX was significantly larger in DOCA-salt than that in DOCA-H2O group (p < 0.001, two-sample t test). However, additional AP5 (PEAQX + AP5) caused minimal Iholding shift in both groups (DOCA-H2O, 0.80 ± 1.42 pA, vs DOCA-salt, 0.77 ± 0.94 pA; Fig. 2A,D). In contrast, ifenprodil (IFD, a selective NR2B antagonist) failed to cause Iholding shift in both DOCA-H2O (F(2,15) = 9; n = 6 neurons from 3 rats; p = 0.93; Bonferroni’s post hoc test following one-way RM-ANOVA) and DOCA-salt (F(2,18) = 33.22; n = 8 neurons from 4 rats; p = 0.99; Bonferroni’s post hoc test following one-way RM-ANOVA), while additional PEAQX uncovered INMDA in both groups (DOCA-H2O, p = 0.01, and DOCA-salt, p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA in both cases). Notably, additional PEAQX (IFD + PEAQX) uncovered larger INMDA in DOCA-salt compared with DOCA-H2O group (DOCA-H2O, 6.80 ± 1.73 pA vs DOCA-salt, 14.27 ± 2.17 pA; p < 0.001; two-sample t test).
Similarly, PPDA (a selective NR2C/2D antagonist) failed to cause Iholding shift in the both DOCA-H2O (F(2,21) = 10.99; n = 8 neurons from 3 rats; p = 0.65; Bonferroni’s post hoc test following one-way RM-ANOVA) and DOCA-salt (F(2,27) = 42.56; n = 10 neurons from 4 rats; p = 0.84; Bonferroni’s post hoc test following one-way RM-ANOVA), while additional PEAQX uncovered INMDA in both groups (DOCA-H2O, p = 0.009, and DOCA-salt, p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA in both cases). Consistently, additional PEAQX (PPDA + PEAQX) uncovered larger INMDA in DOCA-salt compared with DOCA-H2O group (DOCA-H2O, 5.45 ± 1.73 pA vs DOCA-salt, 12.58 ± 1.62 pA; p < 0.001; two-sample t test). These results suggested that Mg2+-sensitive INMDA predominantly represented the activation of NR2A-containing NMDARs in nondepolarized SON MNCs in both DOCA-H2O and DOCA-salt groups.
Decreased EAAT activity in the SON of DOCA-salt rats
EAATs that uptake glutamate, particularly the astrocytic glutamate transporter-1 (GLT1) and glutamate/aspartate transporter (GLAST) isoforms, potently regulate glutamate clearance to maintain ambient glutamate levels in the central nervous system (Fleming et al., 2011; Sun et al., 2014). Increased neurohumoral drive such as heart failure condition-induced glial remodeling caused decreased GLT1 and increased GLAST to elevate ambient glutamate level; thus, increased INMDA of SON NMCs (Potapenko et al., 2012) and similar changes in GLT1 was also observed in SON MNCs during dehydration (Fleming et al., 2011). To know if this is the case in DOCA-salt rats, we compared endogenous EAAT activity by quantifying the magnitude of the INMDA evoked by a EAAT blocker in DOCA-H2O and DOCA-salt rats (Fig. 3).
Comparison of INMDA in the presence of EAAT antagonist, TBOA, in DOCA-H2O and DOCA-salt models. A, Representative current traces showing the effects of TBOA, a glutamate transporter blocker (100 µM), followed by the sequential application of AP5 (200 µM), an NMDA receptor antagonist and kynurenic acid (5 mM), a glutamate receptor antagonist, in holding current. Note that AP5 completely reversed Iholding change induced by TBOA in both DOCA-H2O and DOCA-salt. B, Summarized bar graph showing the mean of holding current change by TBOA (left) and additional AP5 (right). *p < 0.05, compared with DOCA-H2O; n = 6 and 7 in DOCA-H2O and DOCA-salt, respectively.
Bath application of the nonselective EAAT blocker TBOA (100 µM) induced a large inward shift in Iholding (ITBOA) in SON MNCs in both DOCA-H2O (F(2,15) = 54.09; n = 6 neurons from 3 rats; p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA) and DOCA-salt (F(2,18) = 34.57; n = 7 neurons from 3 rats; p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA). Interestingly, TBOA induced inward shift in Iholding (ITBOA) was significantly smaller in DOCA-salt (DOCA-salt, −92.74 ± 14.48 pA) than that in DOCA-H2O group (−164.47 ± 20.92 pA; p = 0.01; two-sample t test). ITBOA was mostly blocked by AP5; thus, the additional application of kynurenic acid, KynA (AP5 + KynA), induced only a minimal Iholding shift in both groups (Fig. 3A). These results suggested that attenuated glutamate transporter activity increased extracellular glutamate concentration, resulting in turn in the activation of a Mg2+-sensitive INMDA in DOCA-salt group.
Exogenous glutamate equalizes INMDA and IPEAQX in DOCA-H2O and DOCA-salt rats
Next, we further tested whether increasing extracellular glutamate levels potentiated the Mg2+-sensitive INMDA in SON MNCs in DOCA-salt group compared with those in DOCA-H2O rats (Fig. 4).
Comparison of INMDA in the presence of exogenous glutamate in DOCA-H2O and DOCA-salt groups. A, Representative current traces show the effect of PEAQX and additional AP5 (100 µM) on the holding current of SON MNCs. Note that PEAQX and AP5 caused similar changes in Iholding. B, Summarized bar graph showing mean of holding current change by PEAQX (left) and additional AP5 (right); n = 7 and 9 in DOCA-H2O and DOCA-salt, respectively.
In the presence of glutamate (10 µM), both PEAQX and additional AP5 (PPDA + AP5) caused significant changes in both DOCA-H2O (F(2,15) = 6.09; p = 0.05; one-way RM-ANOVA, n = 6 neurons from 3 rats) and DOCA-salt (F(2,21) = 12.82; p < 0.001; one-way RM-ANOVA; n = 8 neurons from 3 rats). IPEAQX was not different in DOCA-H2O (31.87 ± 10.89 pA) from DOCA-salt group (33.25 ± 9.3 pA; p = 0.867; two-sample t test). Additional AP5 (PEAQX + AP5) induced similar outward shift in Iholding in both groups (DOCA-H2O, 21.16 ± 10.38 pA vs DOCA-salt, 14.47 ± 4.47 pA; two-sample t test; p = 0.351). As a result, total INMDA was not different in DOCA-H2O (53.03 ± 21.14 pA) and DOCA-salt groups (47.73 ± 12.43 pA; two-sample t test; p = 0.820).
These results suggested that the increased INMDA in DOCA-salt rats was due to blunted GLUT activity, leading to increased levels of endogenous glutamate, but not due to increase or changes in extrasynaptic NMDARs. Moreover, these results indicate that INMDA is mediated by NR2A receptors in both groups.
To determine whether changes in the expression of NMDAR subunits contributed to altered INMDA in DOCA-salt rats, we compared the expression of NR2A-D in DOCA-H2O and the DOCA-salt groups at 1 week PDI. Western blot results showed no significant difference in NR2A-D protein expression between the groups (Fig. 5A,B).
The expression of NMDAR subunit in the DOCA-H2O and DOCA-salt groups. A, Representative image of a Western blot showing NR2A, NR2B, and NR2D subunit expression in DOCA-H2O and DOCA-salt groups. B, Summarized bar graph showing the relative expression of NMDRAs in the SON of DOCA-H2O and DOCA-salt groups. The protein expression was normalized to the level detected in the DOCA-H2O group and compared with the expression in DOCA-salt animals. Summarized data shown are the mean ± SE (n = 3 rats).
Iifenprodil in depolarized SON MNCs did not differ between DOCA-H2O and DOCA-salt rats
We next investigated the sensitivity of INMDA to NR2A, NR2B, and NR2C/D subunit selective antagonists in depolarized neurons (Vholding +40 mV; Fig. 6).
Pharmacology of INMDA in depolarized SON MNCs. A, Representative traces showing the Iholding changes induced by the sequential addition of 1 µM PPDA, 1 µM PEAQX, and 10 µM IFD in DOCA-H2O and DOCA-salt groups with a depolarized membrane potential (Vh, +40 mV). The dotted lines indicate the mean Iholding under each condition. B, Summarized Iholding changes induced by PPDA, PEAQX, and IFD in SON MNCs from both DOCA-H2O and DOCA-salt groups; n = 6 in each group.
In agreement with the previous reports (Fleming et al., 2011; Neupane et al., 2021), IFD caused an inward Iholding shift (Iifenprodil) in depolarized SON MNCs in both DOCA-H2O (F(2,15) = 19.25; n = 6 neurons from 3 rats; p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA) and DOCA-salt (F(2,18) = 32.16; n = 7 neurons from 3 rats; p < 0.001; Bonferroni’s post hoc test following one-way RM-ANOVA). Iifenprodil was not different in DOCA-H2O (26.93 ± 4.46 pA) and DOCA-salt group (26.25 ± 12.74 pA; two-sample t test; p = 0.960). In contrast, PEAQX and PPDA failed to cause significant Iholding changes in both DOCA-H2O (PEAQX, p = 0.96 and PPDA, p = 0.91; Bonferroni’s post hoc test following one-way RM-ANOVA) and DOCA-salt rats (PEAQX, p = 0.95 and PPDA, p = 0.89; Bonferroni’s post hoc test following one-way RM-ANOVA in both cases; Fig. 5C,D). These results suggested that Iifenprodil in depolarized SON MNCs did not contribute to the INMDA mediated by increased ambient glutamate at 1 week PDI.
IPEAQX but not Iifenprodil sensed an increased ambient glutamate concentration in DOCA-salt rats
In the next experiments, we directly investigated the hypothesis that altered ambient glutamate concentration could be sensed by IPEAQX in nondepolarized SON MNCs. For this, we compared the effects of PEAQX on INMDA in the absence and presence of exogenous glutamate (10 µM; Fig. 7).
Mechanism of generating INMDA in the presence of exogenous glutamate in nondepolarized and depolarized SON MNCs. A, Representative current traces show the effect of PEAQX and additional AP5 (100 µM) on the holding current of nondepolarized SON MNCs in basal condition and in the presence of 10 µM glutamate. B, Summarized bar graph showing mean of holding current change by PEAQX (left), additional AP5 (middle), and total INMDA (right); n = 7 and 9 in basal and in the presence of 10 µM, respectively. C, Representative current traces show the effect of PEAQX and additional AP5 (100 µM) on the holding current of depolarized SON MNCs in basal condition and in the presence of 10 µM glutamate. D, Summarized bar graph showing mean of holding current change by PEAQX (left), additional AP5 (middle), and total INMDA (right); n = 6 in both condition. E, Representative current traces show the effect of ifenprodil and additional AP5 (100 µM) on the holding current of depolarized SON MNCs in basal condition and in the presence of 10 µM glutamate. F, Summarized bar graph showing mean of holding current change by IFD (left), additional AP5 (middle), and total INMDA (right); n = 7 and 6 in basal and in the presence of 10 µM, respectively.
As expected, exogenous glutamate significantly increased IPEAQX in nondepolarized SON MNCs from 8.67 ± 1.10 pA (n = 7 neurons from 3 rats) to 31.49 ± 7.71 pA (n = 6 neurons from 3 rats). Additional AP5 (PEAQX + AP5) caused a minimal but significant Iholding shift in the presence of exogenous glutamate (control, 2.06 ± 0.85 pA, n = 7 neurons from 3 rats, vs 10 µM glutamate, 11.37 ± 3.24 pA, n = 6 neurons from 3 rats; p = 0.01 in both cases; two sample t test). As a result, total INMDA in the nondepolarized condition was significantly larger in the presence of 10 µM glutamate (10.73 ± 1.35 pA; n = 6 neurons from 3 rats) compared with the control condition (41.24 ± 10.25 pA, n = 7 neurons from 3 rats; Fig. 7B; p = 0.01; two-sample t test), while the portion of IPEAQX to the total INMDA was not different in the absence and presence of glutamate (82 ± 6.67%, n = 6 vs 76 ± 5.49%, n = 7; p = 0.50; two-sample t test). Noted that IFD did not affect Iholding in nondepolarized SON MNCs even in the presence of exogenous glutamate (control, 1.67 ± 0.95 vs 10 µM glutamate, 1.25 ± 1.21 pA). These results supported the idea that INMDA predominantly represented the activation of NR2A-containing NMDARs in nondepolarized SON MNCs.
We further investigated whether the increased ambient glutamate concentration could be sensed by IPEAQX and Iifenprodil in depolarized SON MNCs. In contrast to IPEAQX in nondepolarized neurons, exogenous glutamate failed to increase IPEAQX of depolarized SON MNCs (control, 2.18 ± 1.98 pA, n = 6 neurons from 3 rats; F(2,15) = 24.59; p = 0.90; Bonferroni’s post hoc test following one-way RM-ANOVA and 10 µM glutamate, 2.20 ± 1.84 pA, n = 6 neurons from 3 rats; F(2,15) = 20.97; p = 0.92; Bonferroni’s post hoc test following one-way RM-ANOVA). Additional AP5 (PEAQX + AP5) uncovered a similar INMDA in both conditions (control, 29.39 ± 5.92 pA and 10 µM glutamate, 6.67 ± 7.25 pA; Fig. 7C,D). As a result, the total INMDA of depolarized SON MNCs was not different in the absence and presence of exogenous glutamate (Fig. 7C,D).
In addition, although IFD caused an inward Iholding shift in depolarized SON MNCs (Figs. 6, 7C,D), exogenous glutamate failed to affect Iifenprodil in depolarized SON MNCs (control, 20.38 ± 2.68 pA, n = 7 neurons from 3 rats vs glutamate, 22 ± 2.90 pA, n = 6 neurons from 3 rats). Additional AP5 (IFD + AP5) uncovered a similar magnitude of INMDA in both conditions (Fig. 7E,F). As a result, total INMDA in depolarized SON MNCs was not different in the absence and presence of exogenous glutamate (Fig. 7E,F) suggesting that membrane depolarization to Vholding of +40 mV maximized INMDA in our recording conditions, thus making it insensible to increased ambient glutamate at 1 week PDI.
Discussion
The main findings of this study are that (1) Mg2+-sensitive INMDA was increased significantly but transiently at post 1 week in DOCA-salt rats; (2) the enhanced IPEAQX is an agreement with attenuated EAAT activity with no changes in NMDAR subunit expression at 1 week PDI; and (3) Iifenprodil in depolarized SON MNCs did not respond to increased ambient glutamate in normal and DOCA-salt groups. These findings indicate that the INMDA in nondepolarized and depolarized SON MNCs is dominantly mediated by NR2A- and NR2B-containing NMDARs, respectively, and the former efficiently sensed the increased ambient glutamate concentration in the SON of normal and hypertensive rats. One limitation of this study is that only male rats were used to avoid the potential hormonal changes in female rats, which directly influence the hypertension pathophysiology. To the best of our knowledge, this is the first evidence that NR2A-containing NMDARs could contribute to tonic excitation mediated by extrasynaptic NMDARs.
INMDA generated by NR2A-containing NMDARs
Glutamate can generate a tonic INMDA when it binds to eNMDARs, while it evokes classical EPSCs via the activation of their synaptic counterparts. Tonic NMDAR current, INMDA, is a hallmark of eNMDAR activity, and NR2A subunit expression is more localized at synaptic sites and exclusively observed in postsynaptic sites (Groc et al., 2006; Paoletti et al., 2013; Cercato et al., 2016; Franchini et al., 2020). Although the presence of NR2A in both sites has been reported (Thomas et al., 2006; Gladding and Raymond, 2011), it was surprising to observe that PEAQX, an NR2A subunit antagonist, blocked INMDA in the present study (Fig. 2). These results suggest that synaptic NR2A-containing NMDARs rather than eNMDARs containing NR2B subunit could generate the Mg2+-sensitive INMDA in nondepolarized SON MNCs. The idea is supported by our results that eNMDAR antagonists including ifenprodil and PPDA failed to affect Iholding of nondepolarized SON MNCs in low Mg2+ condition (Fig. 2). It is noteworthy that the tonic activation of GABAA receptors generating tonic GABAA inhibition were identified superimposed to high-frequency synaptic events (Otis et al., 1991; Salin and Prince, 1996; Hausser and Clark, 1997). Thus, IPEAQX could represent the superimposition of high-frequency synaptic NMDAR currents especially in nondepolarized neurons under low [Mg2+]o conditions. The idea is also in line with the fact that Mg2+ enhances the desensitization of NMDARs (Kampa et al., 2004), while the kinetics of NMDARs are faster at more negative holding potentials (Konnerth et al., 1990; Keller et al., 1991).
Combined with their extrasynaptic location (Lozovaya et al., 2004), NR2B-containing eNMDARs have been known to generate INMDA when exposed to low concentration of ambient glutamate. However, ifenprodil failed to uncover INMDA in nondepolarized SON MNCs even in the presence of exogenous glutamate (Figs. 2, 7). The apparent discrepancy may be reconciled with the idea that Mg2+ unblock, coupled with membrane potential depolarization, is essential to activate NR2B-containing receptors in our recording condition. This idea is in agreement with the fact that ifenprodil uncovered INMDA in depolarized SON MNCs (Figs. 6, 7). Given that NR2A and NR2B confer similar glutamate sensitivity to NMDARs (Erreger et al., 2007; Hansen et al., 2008) and low [Mg2+]o (20 µM) efficiently blocked NMDARs in nondepolarized SON MNCs, these results are also supportive of the idea that EPSCs causing a transient increase of glutamate concentration in the synaptic cleft activates synaptic NR2A receptors to generate IPEAQX in nondepolarized SON MNCs.
However, combined with the fact that EPSC frequency was not different in the DOCA-H2O and DOCA-salt groups (Table 2), the significantly larger IPEAQX in DOCA-salt rats argues against a contribution of superimposed EPSCs mediating IPEAQX. This is further supported by our results showing that increased ambient glutamate equalized IPEAQX in the DOCA-salt and DOCA-H2O groups (Fig. 4). Thus, it is noteworthy that peri- as well as extrasynaptic receptors contribute tonic NMDAR currents (Papouin and Oliet, 2014) as GABAA tonic inhibition (Farrant and Nusser, 2005; Glykys and Mody, 2007; Belelli et al., 2009). Future studies are warranted to investigate whether perisynaptic NMDARs containing NR2A subunit could generate PEAQX-sensitive INMDA in low [Mg2+]o condition and, thus, also respond to an increased ambient glutamate concentration such as in DOCA-salt 1 week.
Phasic current properties of SON MNCs
IPEAQX but not by Iifenprodil contribute to sensing altered ambient glutamate concentrations in DOCA-salt rats
In the present study, NR2A-containing NMDARs generated IPEAQX in nondepolarized SON MNCs in low [Mg2+]o condition, which sensed an increased ambient glutamate, thus generating in turn a larger INMDA in DOCA-salt rats. In contrast, Iifenprodil in depolarized SON MNCs was not different in DOCA-H2O and DOCA-salt groups (Fig. 6). One possible explanation for this difference is that Iifenprodil in depolarized SON MNCs was saturated in our recording condition. This possibility is actually supported by our results showing that exogenous glutamate failed to increase Iifenprodil in depolarized SON MNCs (Fig. 7). This result is also in line with the fact that Iifenprodil was not potentiated following an increase in ambient glutamate concentration in DOCA-salt rats (Fig. 2).
NMDAR phosphorylation reduces the voltage-dependent Mg2+ block of the channels (Chen and Huang, 1992), leading to, as recently shown (Pham et al., 2022), an increase in Iifenprodil. The lack of increase in Iifenprodil in this study suggests that NR2B phosphorylation is not a likely mechanism contributing to changes in NMDAR function reported in DOCA-salt rats. Overall, our results showed that Iifenprodil represents the saturated activity of NR2B-containing NMDARs in depolarized SON MNCs.
Although it is purely speculative, it is interesting to note that “the extrasynaptic space” comprising separate domains was proposed instead of one large homogeneous volume (Papouin and Oliet, 2014). Given that NR2A and NR2B are preferentially located in synaptic and extrasynaptic regions (Kew et al., 1998; Tovar and Westbrook, 1999; Barria and Malinow, 2002), IPEAQX and IIfenprodil may preferentially sense glutamate concentrations in the synaptic cleft over the ambient glutamate concentration in extrasynaptic space, respectively. However, our results showing that exogenous glutamate increased IPEAQX argue against the notion of a functional barrier discriminating “PEAQX-sensitive” synaptic NMDARs and “ifenprodil-sensitive” extrasynaptic regions. Future studies are warranted to identify the spatial component differentiating “PEAQX-sensitive regions” over “ifenprodil-sensitive regions” in the SON.
An increase in ambient glutamate was insufficient to generate a Mg2+-resistant INMDA
In a previous report (Neupane et al., 2021), it was shown that eNMDARs containing homodimeric NR2D subunit were the best candidate generating the Mg2+-resistant INMDA in both nondepolarized and depolarized SON MNCs. However, an “NR2D recall” is essential to generate the Mg2+-resistant INMDA in the matured brain, because NR2D expression is gradually decreased with brain maturation (Monyer et al., 1994; Dunah et al., 1996; Wenzel et al., 1996; Liu and Wong-Riley, 2010). In the present study, DOCA-salt failed to generate an Mg2+-resistant INMDA in SON MNCs, despite the fact that an increase in ambient glutamate concentration was observed at 1 week PDI. Combined with the results that DOCA-salt did not affect NR2D subunit expression (Fig. 5), these results strengthened the idea that “NR2D recall” is essential to generate Mg2+-resistant INMDA in the matured brain (Neupane et al., 2021).
Physiological significance of increased glutamate in SON MNCs in hypertension
Elevated ambient glutamate due to diminished glutamate clearance modifies the excitatory tone that plays a critical role in regulating hypothalamic neurohumoral activation (Potapenko et al., 2012). In hypertension, increased NMDAR-Ca2+ responses upregulate neuronal firing in SON MNCs (Zhang et al., 2017; Zhang and Stern, 2017). In the present study, elevated glutamate in the prodromal stage of hypertension was detected by NR2A subunit-containing NMDARs. Given that INMDA mediated by NR2A-containing NMDARs detected only in the physiological atypical concentration of Mg2+, it may not directly affect neuronal firing in the SON MNCs. However, normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate and that NR2B subunit-containing NMDARs-mediated tonic INMDA is limited and saturated at depolarized SON MNCs (Neupane et al., 2021; Fig. 7), our results showed that INMDA mediated by NR2A-containing NMDARs is an efficient biosensor for detecting altered ambient glutamate level in the brain.
Taken together, our results suggest that synaptic NMDARs containing NR2A subunit generate INMDA in nondepolarized SON MNCs under low Mg2+ condition, while NR2B-containing receptors mediate INMDA in depolarized SON MNCs under normal Mg2+ condition. Thus, IPEAQX represents the tonic activation of NR2A-containing receptors in SON MNCs, standing thus as a useful biomarker for the detection of ambient glutamate concentration in the SON during normal and pathological neurohumoral overdrive.
Footnotes
The authors declare no competing financial interests.
This work was supported by the National Research Foundation of Korea (NRF, 2021R1I1A3051864 and 2023R1A2C100524811 to J.B.P.), the New Faculty Startup Fund from Seoul National University (550-20220057 to J.B.P.), the National Heart, Lung, and Blood Institute Grant (NIH HL090948 to J.E.S.), and the National Institute of Neurological Disorders and Stroke Grant (NIH NS094640 to J.E.S.).
↵*H.S. and R.S. contributed equally to this work.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.