Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus

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.


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 (I NMDA ) 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 I NMDA 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 "Mg 2+ -resistant" I NMDA activated in nondepolarized SON MNCs under the physiological concentration of [Mg 2+ ] 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 Mg 2+ 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 "Mg 2+ -resistant" I NMDA of MNCs in a statedependent manner (Neupane et al., 2021), we investigated whether NR2D-containing eNMDARs contribute to exacerbated I NMDA 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 Mg 2+ -sensitive I NMDA 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 H 2 O group (DOCA-H 2 O) 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).
(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 H 2 O 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 MgCl 2 , 26 NaHCO 3 , 1.2 NaH 2 PO 4 , 10 glucose, and 2.4 CaCl 2 , pH 7.3-7.4,and saturated with 95% O 2 and 5% CO 2 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% O 2 and 5% CO 2 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 I NMDA ) was defined as changes in the holding current (I holding ) in the presence of ionotropic GABA receptor antagonists and was calculated by the difference in I holding measured as the average of a 2-minute steady-state baseline segment obtained before and after the application of NMDAR antagonists.I NMDA was recorded and calculated at −70 mV with low [Mg 2+ ] o (20 µM) or +40 mV with normal aCSF containing 1.2 mM [Mg 2+ ] o unless otherwise stated.Event detection and analysis of spontaneous EPSCs were carried out using MiniAnalysis software (Synaptosoft) at V holding −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.
Statistical analysis.Numerical data are presented as the mean ± SEM.To assess the differences in tonic I NMDA 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 I NMDA was categorized into two modalities: either the Mg 2+ -sensitive tonic I NMDA , measured by I holding shift induced by increasing the extracellular Mg 2+ concentration from 20 μM to 1.2 mM at V h , −70 mV, or the Mg 2+ -resistant tonic I NMDA , measured by I holding shift induced by NMDAR antagonists at V h , −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 I NMDA to various NMDAR antagonists was investigated.To compare I NMDA amplitude and protein expression (NR2A, NR2B, and NR2D) in the H 2 O group and DOCA-salt group, we used one-way ANOVA followed by a post hoc test.

Results
Transient increase of I NMDA 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-H 2 O) 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 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-H 2 O 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-H 2 O groups at 1 week PDI (Table 1).In addition, the urine osmolality was significantly lower in DOCA-salt than that in DOCA-H 2 O rats, meanwhile the serum osmotic pressure was not different in the two groups (Table 1).
In next experiment, we investigated I NMDA in SON MNCs in DOCA-H 2 O and DOCA-salt groups at 1, 2, and 4 weeks PDI.Increasing [Mg 2+ ] o from 20 µM to 1.2 mM induced a significantly larger I holding in DOCA-salt (15.17 ± 2.32 pA, n = 19 neurons from 7 rats) compared with DOCA-H 2 O 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 I holding shift in all tested groups (Fig. 1B,C).These results suggested that more NMDARs could be activated to generate Mg 2+ -sensitive I NMDA in SON MNCs in DOCA-salt rats at 1 week PDI.

Pharmacology of the enhanced I NMDA in DOCA-salt rats
To assess the composition of NMDARs that potentiate Mg 2+ -sensitive I NMDA at 1 week PDI, we investigated I NMDA at V holding −70 mV in low Mg 2+ aCSF and compared its sensitivity with NR2A, NR2B, and NR2C/D subunit selective antagonists in DOCA-H 2 O and DOCA-salt group (Fig. 2).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 I NMDA 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 I NMDA evoked by a EAAT blocker in DOCA-H 2 O and DOCA-salt rats (Fig. 3).Bath application of the nonselective EAAT blocker TBOA (100 µM) induced a large inward shift in I holding (I TBOA ) in SON MNCs in both DOCA-H 2 O (F (2,15) = 54.09;n = 6 neurons from 3 rats; p < 0.001; Bonferroni's post hoc test following oneway 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 I holding (I TBOA ) was significantly smaller in DOCA-salt (DOCA-salt, −92.74 ± 14.48 pA) than that in DOCA-H 2 O group (−164.47 ± 20.92 pA; p = 0.01; two-sample t test).I TBOA was mostly blocked by AP5; thus, the additional application of kynurenic acid, KynA (AP5 + KynA), induced only a minimal I holding 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 Mg 2+ -sensitive I NMDA in DOCA-salt group.
Exogenous glutamate equalizes I NMDA and I PEAQX in DOCA-H 2 O and DOCA-salt rats Next, we further tested whether increasing extracellular glutamate levels potentiated the Mg 2+ -sensitive I NMDA in SON MNCs in DOCA-salt group compared with those in DOCA-H 2 O rats (Fig. 4).
These results suggested that the increased I NMDA 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 I NMDA is mediated by NR2A receptors in both groups.
To determine whether changes in the expression of NMDAR subunits contributed to altered I NMDA in DOCA-salt rats, we compared the expression of NR2A-D in DOCA-H 2 O 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).

I ifenprodil in depolarized SON MNCs did not differ between DOCA-H 2 O and DOCA-salt rats
We next investigated the sensitivity of I NMDA to NR2A, NR2B, and NR2C/D subunit selective antagonists in depolarized neurons (V holding +40 mV; Fig. 6).
In agreement with the previous reports (Fleming et al., 2011;Neupane et al., 2021), IFD caused an inward I holding shift (I ifenprodil ) in depolarized SON MNCs in both DOCA-H 2 O (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).I ifenprodil was not different in DOCA-H 2 O (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 I holding changes in both DOCA-H 2 O (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 I ifenprodil in depolarized SON MNCs did not contribute to the I NMDA mediated by increased ambient glutamate at 1 week PDI.I PEAQX but not I ifenprodil 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 I PEAQX in nondepolarized SON MNCs.For this, we compared the effects of PEAQX on I NMDA in the absence and presence of exogenous glutamate (10 µM; Fig. 7).As expected, exogenous glutamate significantly increased I PEAQX 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 I holding 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 I NMDA 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 I PEAQX to the total I NMDA 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 I holding 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 I NMDA predominantly represented the activation of NR2A-containing NMDARs in nondepolarized SON MNCs.
In addition, although IFD caused an inward I holding shift in depolarized SON MNCs (Figs. 6, 7C,D), exogenous glutamate failed to affect I ifenprodil 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 I NMDA in both conditions (Fig. 7E,F).As a result, total I NMDA in depolarized SON MNCs was not different in the absence and presence of exogenous glutamate (Fig. 7E,F) suggesting that membrane depolarization to V holding of +40 mV maximized I NMDA 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) Mg 2+ -sensitive I NMDA was increased significantly but transiently at post 1 week in DOCA-salt rats; (2) the enhanced I PEAQX is an agreement with attenuated EAAT activity with no changes in NMDAR subunit expression at 1 week PDI; and (3) I ifenprodil in depolarized SON MNCs did not respond to increased ambient glutamate in normal and DOCA-salt groups.These findings indicate that the I NMDA 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.

I NMDA generated by NR2A-containing NMDARs
Glutamate can generate a tonic I NMDA when it binds to eNMDARs, while it evokes classical EPSCs via the activation of their synaptic counterparts.Tonic NMDAR current, I NMDA , 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 I NMDA in the present study (Fig. 2).These results suggest that synaptic NR2A-containing NMDARs rather than eNMDARs containing NR2B subunit could generate the Mg 2+ -sensitive I NMDA in nondepolarized SON MNCs.The idea is supported by our results that eNMDAR antagonists including ifenprodil and PPDA failed to affect I holding of nondepolarized SON MNCs in low Mg 2+ condition (Fig. 2).It is noteworthy that the tonic activation of GABA A receptors generating tonic GABA A inhibition were identified superimposed to high-frequency synaptic events (Otis et al., 1991;Salin and Prince, 1996;Hausser and Clark, 1997).Thus, I PEAQX could represent the superimposition of high-frequency synaptic NMDAR currents especially in nondepolarized neurons under low [Mg 2+ ] o conditions.The idea is also in line with the fact that Mg 2+ 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 I NMDA when exposed to low concentration of ambient glutamate.However, ifenprodil failed to uncover I NMDA in nondepolarized SON MNCs even in the presence of exogenous glutamate (Figs. 2, 7).The apparent discrepancy may be reconciled with the idea that Mg 2+ 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 I NMDA 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 [Mg 2+ ] 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 I PEAQX in nondepolarized SON MNCs.
However, combined with the fact that EPSC frequency was not different in the DOCA-H 2 O and DOCA-salt groups (Table 2), the significantly larger I PEAQX in DOCA-salt rats argues against a contribution of superimposed EPSCs mediating I PEAQX .This is further supported by our results showing that increased ambient glutamate equalized I PEAQX in the DOCA-salt and DOCA-H 2 O groups (Fig. 4).Thus, it is noteworthy that peri-as well as extrasynaptic receptors contribute tonic NMDAR currents (Papouin and Oliet, 2014) as GABA A 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 I NMDA in low [Mg 2+ ] o condition and, thus, also respond to an increased ambient glutamate concentration such as in DOCA-salt 1 week.I PEAQX but not by I ifenprodil contribute to sensing altered ambient glutamate concentrations in DOCA-salt rats In the present study, NR2A-containing NMDARs generated I PEAQX in nondepolarized SON MNCs in low [Mg 2+ ] o condition, which sensed an increased ambient glutamate, thus generating in turn a larger I NMDA in DOCA-salt rats.In contrast, I ifenprodil in depolarized SON MNCs was not different in DOCA-H 2 O and DOCA-salt groups (Fig. 6).One possible explanation for this difference is that I ifenprodil 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 I ifenprodil in depolarized SON MNCs (Fig. 7).This result is also in line with the fact that I ifenprodil was not potentiated following an increase in ambient glutamate concentration in DOCA-salt rats (Fig. 2).
NMDAR phosphorylation reduces the voltage-dependent Mg 2+ block of the channels (Chen and Huang, 1992), leading to, as recently shown (Pham et al., 2022), an increase in I ifenprodil .The lack of increase in I ifenprodil 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 I ifenprodil 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), I PEAQX and I Ifenprodil 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 I PEAQX 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 Mg 2+ -resistant I NMDA In a previous report (Neupane et al., 2021), it was shown that eNMDARs containing homodimeric NR2D subunit were the best candidate generating the Mg 2+ -resistant I NMDA in both nondepolarized and depolarized SON MNCs.However, an "NR2D recall" is essential to generate the Mg 2+ -resistant I NMDA 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 Mg 2+ -resistant I NMDA 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 Mg 2+ -resistant I NMDA 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-Ca 2+ 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 I NMDA mediated by NR2A-containing NMDARs detected only in the physiological atypical concentration of Mg 2+ , 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 I NMDA is limited and saturated at depolarized SON MNCs (Neupane et al., 2021;Fig. 7), our results showed that I NMDA 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 I NMDA in nondepolarized SON MNCs under low Mg 2+ condition, while NR2B-containing receptors mediate I NMDA in depolarized SON MNCs under normal Mg 2+ condition.Thus, I PEAQX 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.

Figure 1 .
Figure 1.Increased I NMDA of SON MNCs in DOCA-salt rats.A, Systolic blood pressure at different times in naive control (normal control, n = 6), DOCA-H 2 O (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 Mg 2+ , followed by the sequential application of AP5 (200 µM), a NMDA receptor antagonist, in holding current in DOCA-H 2 O and DOCA-salt model rats at 1 week.Representative current traces show the effect of sequential application of Mg 2+ (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 Mg 2+ (C) and additional AP5 (D) in each week, respectively.**p < 0.01, compared with DOCA-H 2 O.

Figure 2 .
Figure 2. Pharmacology of I NMDA in DOCA-H 2 O 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 I holding , while additional PEAQX caused larger I holding changes in DOCA-salt groups.D, Summarized bar graph showing mean of holding current change by each antagonist.*p < 0.05, compared with DOCA-H 2 O; n = 6 and 8 in DOCA-H 2 O and DOCA-salt, respectively.

Figure 3 .
Figure 3.Comparison of I NMDA in the presence of EAAT antagonist, TBOA, in DOCA-H 2 O 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 I holding change induced by TBOA in both DOCA-H 2 O 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-H 2 O; n = 6 and 7 in DOCA-H 2 O and DOCA-salt, respectively.

Figure 4 .
Figure 4. Comparison of I NMDA in the presence of exogenous glutamate in DOCA-H 2 O 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 I holding .B, Summarized bar graph showing mean of holding current change by PEAQX (left) and additional AP5 (right); n = 7 and 9 in DOCA-H 2 O and DOCA-salt, respectively.

Figure 5 .
Figure 5.The expression of NMDAR subunit in the DOCA-H 2 O and DOCA-salt groups.A, Representative image of a Western blot showing NR2A, NR2B, and NR2D subunit expression in DOCA-H 2 O and DOCA-salt groups.B, Summarized bar graph showing the relative expression of NMDRAs in the SON of DOCA-H 2 O and DOCA-salt groups.The protein expression was normalized to the level detected in the DOCA-H 2 O group and compared with the expression in DOCA-salt animals.Summarized data shown are the mean ± SE (n = 3 rats).

Figure 6 .
Figure 6.Pharmacology of I NMDA in depolarized SON MNCs.A, Representative traces showing the I holding changes induced by the sequential addition of 1 µM PPDA, 1 µM PEAQX, and 10 µM IFD in DOCA-H 2 O and DOCA-salt groups with a depolarized membrane potential (Vh, +40 mV).The dotted lines indicate the mean I holding under each condition.B, Summarized I holding changes induced by PPDA, PEAQX, and IFD in SON MNCs from both DOCA-H 2 O and DOCA-salt groups; n = 6 in each group.

Figure 7 .
Figure 7. Mechanism of generating I NMDA 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 I NMDA (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 I NMDA (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 I NMDA (right); n = 7 and 6 in basal and in the presence of 10 µM, respectively.

Table 1 .
Changes in metabolic parameters after 1 week in DOCA-salt induced hypertensive model

Table 2 .
Phasic current properties of SON MNCs