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Iranian Journal of Basic Medical Sciences logoLink to Iranian Journal of Basic Medical Sciences
. 2021 May;24(5):586–594. doi: 10.22038/ijbms.2021.53181.11978

Role of the glutamatergic system of ventrolateral periaqueductal gray (vlPAG) in the cardiovascular responses in normal and hemorrhagic conditions in rats

Vida Alikhani 1, Reza Mohebbati 2, Mahmoud Hosseini 3, Abolfazl Khajavirad 1, Mohammad Naser Shafei 1,2,*
PMCID: PMC8244607  PMID: 34249259

Abstract

Objective(s):

Periaqueductal gray (PAG) is a mesencephalic area divided into four columns including ventrolateral periaqueductal gray (vlPAG). vlPAG plays a role in cardiovascular regulation during normal and hemorrhagic (Hem) conditions. Due to presence of glutamate in this area, we evaluated the effect of glutamatergic receptors of this area on cardiovascular activity in normotensive and hypovolemic Hem rats.

Materials and Methods:

Animals were divided into twelve groups: saline (vehicle), Glutamate, GYK52466 (non-NMDA receptor antagonist), and MK801 (NMDA receptor antagonist) with and without Glu microinjected into vlPAG in normal and Hem conditions. Following the femoral artery cannulating and microinjecting, changes (Δ) of heart rate (HR), systolic blood pressure (SBP), and mean arterial pressure (MAP) were recorded via a PowerLab unit.

Results:

In normotensive conditions, microinjection of Glu increased ΔMAP, ΔSBP, and ΔHR (P<0.001). MK-801 and GYKI-52466 nonsignificant reduced cardiovascular responses than vehicle while their changes were significant compared with glutamate (P<0.001). Co-injection of GYKI- 52466 with Glu did not significantly reduce ΔSBP and ΔMAP induced by Glu (P>0.05) but co-injection of MK-801 with Glu significantly attenuate these effects(P<0.01). In Hem, Glu increased ΔSBP, ΔMAP, and ΔHR (P<0.05). GYKI-52466 alone did not change cardiovascular responses but MK-801 decreased ΔSBP than Hem (P<0.01). Co-injection of GYKI-52466 with Glu had significant(P<0.05) but MK-801 with Glu had no significant effect compared with Hem (P>0.05).

Conclusion:

The glutamatergic system of vlPAG increases cardiovascular values that are mostly mediated through the NMDA receptor. Since vlPAG is well known as an inhibitory region, it seems that glutamate does not have a noteworthy cardiovascular role in vlPAG during Hem and normal conditions.

Key Words: Blood pressure, Cardiovascular system, Glutamic acid, Hemorrhage, Periaqueductal gray

Introduction

Periaqueductal gray (PAG) is a mesencephalic structure described as being related to the neuronal pathways that affect cardiovascular regulation and autonomic function (1). PAG divides into four longitudinal columns anatomically: dorsomedial (dmPAG), dorsolateral (dl PAG), lateral (lPAG), and ventrolateral (vlPAG) columns (2). VlPAG has various functions including, fear and defensive behavior (3), food intake (4), rapid eye movement (REM), sleep regulation (5), and involvement in pain modulation (6). VlPAG projects to numerous cardiovascular regulating areas such as the rostral ventromedial medulla (RVLM), an important cardiovascular regulation region. (7). Some studies show that chemical stimulation of vlPAG decreases blood pressure (BP) and heart rate (HR) in rats (1, 7). VlPAG is also related to the nucleus tractus solitarius (NTS), a crucial area for baroreflex and chemoreflex control. There is evidence that vlPAG could modulate tachycardia followed by baroreflex activation (1, 8).

Various neurotransmitters such as acetylcholine, norepinephrine, and glutamate (Glu) are found in vlPAG, which cause different responses in BP and HR (1). It has been reported that vlPAG neurons are activated during hemorrhage, and inactivation of vlPAG can cause hypotension and hemorrhagic bradycardia (9). Glutamate through metabotropic and ionotropic (divide into N-methyl-d-aspartic acid (NMDA) and non- N-methyl-d-aspartic acid (non-NMDA) receptor subtypes) receptors plays a crucial role in regulating the cardiovascular central system (10). Some studies depict the role of glutamate receptors in hemorrhage and blood pressure (11-13). Furthermore, Glu and its ionotropic receptors (NMDA and non-NMDA) in vlPAG nucleus have been reported (11, 14). The blockade of NMDA glutamate receptor in vlPAG inhibits cardiovascular responses induced by the lateral hypothalamic area (11, 15). The aim and novelty of the current study is evaluation of the Glut receptor types involved in the cardiovascular responses during normal and hemorrhage conditions in vlPAG nucleus. Also, in this study, it is significant whether excitation of Glut projection from vlPAG to RVLM directly or indirectly has a role in improvement of HEM condition.

Materials and Methods

Animals

Seventy-two male Wistar rats (250–290 g) were delivered from the animal house of the Medicine Faculty at Mashhad University of Medical Sciences. The rats were housed under standard conditions with ad libitum feeding and water, under a 12-hr light/dark cycle. The experimental process was conducted in accordance with the University Ethical Committee guidelines (approval ID: IR.MUMS.MEDICAL.REC.1398.338).

Drugs

Glutamate (Glu), the principal excitatory neurotransmitter in the central nervous system, GYKI-52466 (1-(4-aminophenyl)-4 methyl-7, 8-methylenedioxy-5H-2, 3-benzodiazepine) (GYK, a selective non-competitive AMPA (non-NMDA) receptor antagonist), MK-801 (MK, a selective non-competitive NMDA antagonist), and urethane, as an anesthetic, were used in this study (16, 17). All drugs were purchased from Sigma Aldrich Chemical Co., USA.

Animal cannulation and cardiovascular response measurement

At first, the rats were anesthetized deeply with urethane (1.5 g/kg). The femoral artery was then cannulated with a heparinized angiocath catheter (22-gauge) for recording cardiovascular parameters and withdrawing blood by connected syringe (18). The angiocath catheter was connected to a blood pressure transducer attached to a PowerLab system (ID instrument, Australia). BP and HR were recorded by the PowerLab system.

During the study, the animal’s body temperature was maintained at 37.5 °C with a warmer throughout the experiment.

Stereotaxic and drug microinjection

After arterial cannulation, the animal was mounted on the stereotactic frame, and the head was fixed. VlPAG area’s coordination was determined based on Paxinos and Watson rat brain atlas (AP: 6.6–8.7 mm, L: ±0–1.5 and H: 5.5–6.5 mm) (19). Then, a hole about 2 mm in diameter was drilled into the skull, and drugs were microinjected into vlPAG using a micropipette with 35–40 µm diameter (Stoelting, USA) connected to a syringe and attached to a manual injector (Stoelting, USA) (20).

Animals groups

The animals were divided into two main groups, including (A) normotensive and (B) hypotensive hemorrhagic (Hem), then subdivided into the following subgroups (n=6):

A) Normotensive groups: 1) vehicle (saline), 2) Glu, 3) GYK, 4) Co-injection of GYK + Glu, 5) MK, and 6) Co-injection of MK + Glu

B) Hemorrhage (Hem) groups: 1) vehicle, 2) Glu, 3) GYK, 4) GYK + Glu, 5) MK, and 6) MK + Glu were microinjected into vlPAG.

Doses of drugs in all groups for Glu, GYK, and MK were 50 nmol, 300 nmol, and 0.5 nmol, respectively (21-23). The microinjection volume for all drugs was 100–150 nl (18).

Hemorrhage protocol

In Hem groups, after stabilization of cardiovascular parameters (approximately 5 min), about 15% of Total Blood Volume (TBV) was withdrawn during ten minutes (5th min to 15th min) from the femoral artery cannula (18). Hem was induced before microinjection. TBV was calculated according to this equation: 0.06 ml per gr (Body Weight)× Body Weight +0.77 (24). This volume (15%) could reduce about 30 mmHg of SBP that appropriate conditions to assess central cardiovascular areas involved in Hem (24). At the end of the experiment, animals were sacrificed by an overdose of urethane. The brains were removed from skulls and kept for 24 hr in 10% formalin for tissue fixation; next, a vibratome was used to cut thin slices with 60-micron thickness. The slides were observed under a light microscope for verification of the microinjection site, according to atlas of Paxinos and Watson (25).

Data analysis

The data were expressed as mean±SEM. Cardiovascular variables, including MAP, HR, and SBP were recorded, and their changes (Δ) were calculated to evaluate the trend of changes several times. Analysis of this data was done by repeated-measures ANOVA, followed by a post-hoc Tukey’s test. Moreover, peak changes of ΔSBP, ΔMAP, and ΔHR were analyzed using (one-way ANOVA and Tukey’s post hoc test). P<0.05 was considered significant.

Results

Effect of saline microinjected into vlPAG nucleus on cardiovascular responses in normotensive rats

In this group, cardiovascular responses before and after microinjection of saline were examined. Before saline microinjection, the cardiovascular responses for MAP, SBP, and HR were 113.34±12.5 mmHg, 135.9 ± 11.16 mmHg, and 384.8 ± 17.22 beats/min, respectively. However, microinjection of saline did not significantly change those parameters (MAP: 110.4±10.6 mmHg, SBP: 131.3±9.2 mmHg, and HR: 379.5 ± 14.6 beats/min).

Effect of glutamate, GYK, and MK microinjected into vlPAG nucleus on cardiovascular responses in normotensive rats

To determine the cardiovascular effects, Glu, GYK, and MK were microinjected into vlPAG, and cardiovascular changes were evaluated. Microinjection of Glu alone increased ∆SBP, ∆MAP, and ∆HR compared with the vehicle group (P<0.001, Figure 1 parts A, B, and C, respectively). Microinjection of GYK and MK alone did not change the cardiovascular parameters compared with the vehicle group over time (repeated measures ANOVA, P>0.05) while their co-injections with Glu attenuated the Glu response. ∆SBP and ∆MAP in GYK + Glu showed significant differences compared with the vehicle group (P<0.01). However, the effect of MK + Glu on cardiovascular parameters did not significantly change (P>0.05) compared with the vehicle group.

Figure 1.

Figure 1

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Time course of ∆SBP (A), ∆MAP(B), and ∆HR (C), after microinjection of saline, glutamate, and NMDA (MK) and non-NMDA antagonist (GYK) of glutamate receptor into vlPAG nucleus. Data were expressed as mean±SEM; n= 6 (repeated measures ANOVA). ∆MAP: mean arterial pressure, ∆SBP: systolic blood pressure, ∆HR: heart rate, vehicle: saline microinjection, Glu: glutamate, GYK: GYKI-52466, MK: MK801. ***: P<0.001, **: P<0.01, and *: P<0.05 vs vehicle group, ###:P<0.001, ##: P<0.01, and #: P<0.05 vs Glu group

Time-course changes of ∆SBP, ∆MAP, and ∆HR in Glu, GYK, and MK groups have also been shown in Figure 1. The difference of GYK and MK in ∆SBP, ∆MAP, and ∆HR was significant compared with the Glu group over time (repeated measures ANOVA, P<0.001, Figure 1). ∆SBP and ∆MAP differences in co-injection of GYK + Glu and MK + Glu groups did not significantly change (P>0.05, Figures 1 A and B), and only ∆HR decreased significantly compared with the Glu group (P<0.001, Figure 1 C).

Glu microinjection into vlPAG significantly increased all cardiovascular parameters’ peak changes compared with the vehicle group (P<0.001, Figure 2). Also, GYK + Glu enhanced the peak change of ∆SBP, ∆MAP, and ∆HR compared with the vehicle group (P<0.05 to P<0.001, Figure 2). MK + Glu microinjection increased ∆SBP (P<0.01, Figure 2 A), and MK alone decreased the peak changes in ∆HR compared with the vehicle group (P<0.01, Figure 2 C).

Figure 2.

Figure 2

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Peak changes of ∆SBP (a), ∆MAP(b), and ∆HR (c), after microinjection of saline, glutamate, NMDA (MK), and non-NMDA antagonist (GYK) of glutamate receptor and their co-injection with Glu into vlPAG nucleus. Data were expressed as mean±SEM; n= 6 (one-way ANOVA). ∆MAP: mean arterial pressure, ∆SBP: systolic blood pressure, ∆HR: heart rate, vehicle: saline microinjection, Glu: glutamate, GYK: GYKI-52466, MK: MK801. ***: P<0.001, **: P<0.01, and *: P<0.05 vs vehicle group, ###: P<0.001, ##: P<0.01, and #: P<0.05 vs Glu group

GYK and MK microinjection alone and co-injection of MK + Glu significantly decreased the peak changes of vascular parameters compared with the Glu group (P<0.05 to P<0.001, Figure 2), and co-injection of GYK + Glu just decreased the peak change of ∆HR compared with the Glu group (P<0.001, Figure 2 C).

Effect of glutamate, GYK, and MK microinjected into vlPAG nucleus on cardiovascular responses in hemorrhagic rats

In the current experiment, to investigate the role of glutamatergic neurons of vlPAG in hypovolemic hypotension condition, 5 min after Hem, Glu, GYK, and MK alone and together were microinjected into vlPAG, cardiovascular responses were evaluated. Time-course changes of ∆SBP, ∆MAP, and ∆HR in the Hem groups treated with Glu, GYK, and MK are shown in Figures 3 and 4, separately. As it has been indicated, Hem induction caused a significant decrease in ∆SBP and ∆MAP compared with the vehicle group over time (repeated measures ANOVA, P<0.05 to P<0.01), and ∆HR increased, but it was not significant (P>0.05). Microinjection of Glu into vlPAG ameliorates the hypotensive responses induced by Hem over time (repeated measures ANOVA, P<0.05, Figure 3). ∆SBP, ∆MAP, and ∆HR induced by Hem did not change after microinjection of GYK alone and co-injection with Glu compared (P>0.05, Figure 3). ∆SBP changes in the MK alone group significantly decreased compared with the Hem group (P<0.01, Figure 4 A). Co-injection of MK + Glu did not change the cardiovascular responses compared with the Hem group (P>0.05, Figure 4).

Figure 3.

Figure 3

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Time course of ∆SBP (A), ∆MAP(B), and ∆HR (C) after microinjection of saline, glutamate, GYK the non-NMDA antagonist of glutamate receptor, and co-injection of GYK and Glu into vlPAG nucleus in hemorrhagic condition. Data were expressed as mean±SEM; n= 6 (repeated measures ANOVA). ∆MAP: Mean arterial pressure, ∆SBP: Systolic blood pressure, ∆HR: Heart rate, vehicle: saline microinjection, Glu: glutamate, GYK: GYKI-52466. Differences with P-value <0.05 were considered significant. ***: P<0.001, **: P<0.01, and *: P<0.05 vs vehicle group, ###: P<0.001, ##: P<0.01, and #: P<0.05 vs Hem+saline group and ^^^: P<0.001, ^^: P<0.01, and ^: P<0.05 vs Glu group

Figure 4.

Figure 4

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Time course of ∆SBP (A), ∆MAP(B), and ∆HR (C), after microinjection of the saline, glutamate, MK the NMDA antagonist of glutamate receptor, and co-injection of MK and Glu into vlPAG nucleus in hemorrhagic condition. Data were expressed as mean±SEM; n= 6 (repeated measures ANOVA). ∆MAP: mean arterial pressure, ∆SBP: systolic blood pressure, ∆HR: heart rate, vehicle: saline microinjection, Glu: glutamate, GYK: GYKI-52466, MK: MK801. Differences with P-value <0.05 were considered significant. ***: P<0.001, **: P<0.01, and *: P<0.05 vs vehicle group, ###: P<0.001, ##: P<0.01, and #: P<0.05 vs Hem+saline group and ^^^: P<0.001, ^^: P<0.01, and ^: P<0.05 vs Glu group

Co-injection of MK + Glu significantly reduced ∆SBP and ∆MAP with respect to the Glu group over time (repeated measures ANOVA, P<0.05 to P<0.01, Figures 4 A and B). GYK + Glu effects on ∆SBP, ∆MAP, and ∆HR compared with the Glu group were not significant (P>0.05, Figure 3). GYK microinjection decreased ∆SBP and ∆MAP compared with the Glu group over time (repeated measures ANOVA, P<0.05 to P<0.001 Figures 3 A and B), and MK microinjection significantly decreased the cardiovascular responses compared with the Glu group (P<0.001, Figures 3 and 4, parts A, B, and C).

Hem significantly increased the peak changes of ∆HR compared with the vehicle group (P<0.01 Figure 5 C). The peak change of ∆SBP and ∆MAP non-significantly decreased (P>0.05, Figures 5 A and B). Glu microinjection increased the peak changes of ∆SBP, ∆MAP, and ∆HR compared with the vehicle group (P<0.01 to P<0.001, Figure 5).

Figure 5.

Figure 5

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Peak changes of ∆SBP (A), ∆MAP (B), and ∆HR (C) after microinjection of saline, glutamate, NMDA (MK), and non-NMDA antagonist (GYK) of glutamate receptor and co-injection of them with Glu into vlPAG nucleus in hemorrhagic condition. Data were expressed as mean±SEM; n=6 (one-way ANOVA). ∆MAP: mean arterial pressure, ∆SBP: systolic blood pressure, ∆HR: heart rate, vehicle: saline microinjection, Glu: glutamate, GYK: GYKI-52466, MK: MK801. Differences with P-value<0.05 were considered significant. ***: P<0.001, **: P<0.01, and *: P<0.05 vs vehicle group, ###: P<0.001, ##: P<0.01, and #: P<0.05 vs Hem+saline group and ^^^: P<0.001, ^^: P<0.01, and ^: P<0.05 vs Glu group

The peak changes of the vascular parameters showed that hypotension induced by Hem improved by microinjection of Glu (P<0.001, Figure 5), and the peak changes of ∆MAP and ∆SBP were ameliorated by co-injection of GYK + Glu (P<0.05), with no significant effect on ∆HR (P>0.05, Figure 5). None the peak changes of vascular parameters were affected by GYK alone (P>0.05, Figure 5) compared with the Hem group. The peak changes of ∆SBP, ∆MAP, and ∆HR were significantly changed by MK (P<0.001, Figure 5), but MK + Glu’s co-injection did not alter the peak changes of ∆SBP, ∆MAP, and ∆HR in comparison with the Hem group (P>0.05).

The peak changes of ∆SBP, ∆MAP, and ∆HR in GYK, MK, and MK + Glu groups significantly decreased compared with the Glu group (P<0.01 to P<0.001, Figure 5). Co-injection of GYK + Glu did not cause a significant difference in ∆SBP and ∆MAP compared with the Glu group (P>0.05), and only peak changes of ∆HR significantly decreased (P<0.001, Figure 5 part C).

Discussion

According to the present study, in normotensive rats, microinjection of Glu into vlPAG increased cardiovascular responses, and these effects were mostly mediated by the NMDA receptor, while non-NMDA antagonists did not affect cardiovascular responses. Co-injection of Glu with NMDA and non-NMDA receptor antagonist attenuated all effects of Glu.

VlPAG has been revealed to be involved in cardiovascular regulation (11). Type of vlPAG projections is unknown, as some reports show that stimulation of vlPAG causes excitatory output from vlPAG (7). Despite that, some recent studies have shown that chemical stimulation of vlPAG causes depressor responses and a decrease in the heart rate (26, 27). It seems to contradict our findings, since Glu microinjection into vlPAG has shown pressor responses. One hypothesis is that the hypotensive effect of vlPAG was not mediated by Glu. Another possible suggestion is attributed to the consciousness condition, as we evaluated anesthetized rats, while that finding might be due to different conditions or methods, for instance, in unanesthetized decerebrate animals (28).

Ionotropic receptors of Glu are subdivided into two groups: NMDA and non-NMDA (AMPA and kainate) receptors, which affect several brain functions such as the learning process (29), control neuronal excitability (30), neural plasticity (31), and also autonomic responses (32). The central cardiovascular regulation of the NMDA receptors was also revealed in several brain regions such as RVLM, paraventricular nucleus (PVN), PBN, NTS, and vlPAG. These studies have reported that the Glu via NMDA and non-NMDA receptors can significantly increase the cardiovascular parameters in the mentioned nuclei (33-36). The observed results were also in line with the findings of these studies and showed that microinjection of the Glu into vlPAG significantly increased the cardiovascular responses in the normotensive rats. In this condition, the antagonist of the non-NMDA receptor in vlPAG had no remarkable effect on BP. The presence of both NMDA and non-NMDA receptors has been reported in vlPAG (14). It is known that Glu can activate all types of ionotropic-Glu receptors (37). Since the non-NMDA receptor in vlPAG did not demonstrate a considerable effect on cardiovascular regulation, it is suggested that NMDA was an essential receptor in vlPAG, which was involved in cardiovascular regulation. Consistent with these results, we also reported in our previous study that the NMDA receptor was the primary receptor in cardiovascular regulation in the CnF (20).

The excitatory effects of NMDA receptors are mediated by Glu and glycine receptor binding and channel permeability to Ca2+ conductance (38). Therefore, it was suggested that Glu in vlPAG had an excitatory effect on the cardiovascular system by the mentioned mechanism. On the other hand, it is shown that there are different neuron populations in vlPAG, including glutamatergic, GABAergic, dopaminergic, and serotonergic neurons (39). Since MK microinjection into vlPAG decreased BP and HR, it was inferred that Glu was released in normal conditions and activated an excitatory projection in vlPAG via NMDA receptor and increased the cardiovascular responses.

vlPAG is a mesencephalic nucleus that has a great connection with the other brain regions associated with cardiovascular regulation, such as the caudal midline medulla (CMM), RVLM, CVLM, NTS, and the Cuneiform nucleus (CnF) (18, 40). So, it is reasonable that the cardiovascular effect of Glu is mediated via connection of vlPAG with the aforementioned areas. There is evidence that some of the pathways associated with the cardiovascular impact of vlPAG are indirect and mostly mediated via RVLM, which is a considerable important sympathoexcitatory region in the medulla (41).

Arterial pressure and vasoconstrictor tone are associated with sympathetic pathways to the heart and arteries, and it is known that the destination of the preganglionic sympathetic neurons is the intermediolateral column (IML) which has an essential role in the mediation of vlPAG-evoked cardiovascular responses (28). Since the direct projections from vlPAG to IML are unknown, it indicates indirect reaches to the IML through synapses in the pons or the medulla (28).

VlPAG-CVLM-RVLM pathway is reported (40), and it is known that there are glutamatergic projections from vlPAG (14) to GABAergic neurons in the CVLM projecting to RVLM (40). In the caudal medulla, there are probably many more than six distinct areas which excitatory amino acids (EAA) microinjection stimulate alteration (decrease or increase) in arterial blood pressure (40). The pressor effect induced by Glu microinjection into vlPAG could be mediated by the interaction of Glu and GABAergic neurons in the CVLM, as Glu causes disinhibition in GABAergic neurons of CVLM, so RVLM would not be affected by GABA, and therefore RVLM activation causes pressor responses. According to this evidence, it was suggested that Glu in vlPAG could decrease the activity of RVLM vasomotor neurons via disinhibition of GABAergic projections to RVLM.

VlPAG-CMM-RVLM pathway has been described as involved in cardiovascular regulation through glutamatergic projections to CMM (14, 42). It is known that there is a GABAergic-glutamatergic neural circuit in vlPAG, and activation or inhibition of each neural group can affect Glu neurons projecting to RVLM (43). CMM encompasses caudal raphe nuclei that its serotonergic neurons project to RVLM (41).

Due to the involvement of CMM and RVLM in cardiovascular regulation (40, 44), it was suggested that microinjecting Glu into vlPAG caused the interaction of Glu in vlPAG and serotoninergic neurons of CMM. So, it gives rise to disinhibition of pressor responses of RVLM. Although, the exact neurotransmitters involved are not clear and more studies are prerequisites for proving these suggestions.

It is well known that raphe nuclei are involved in cardiovascular responses (45). Moreover, the excitatory afferents from vlPAG to nucleus raphe magnus (NRM) (46) and the rostral half of the nucleus raphe obscurus (NRO) are indicated (47). Mediation of vlPAG-raphe nuclei occurs with Glu (48). Hence, it is likely that glutamatergic projections from vlPAG could regulate cardiovascular activation via NRM or NRO. On the other hand, it has been shown that stimulation of these nuclei evokes inhibitory neurons projecting to RVLM (47). The possible hypothesis is that NRM/NRO-RVLM pathways are not monosynaptic, and RVLM stimulation is mediated indirectly via inhibitory interneurons. Therefore, activation of NRM or NRO neurons gives rise to disinhibition of RVLM and increasing BP.

VlPAG also projects to NTS, the region for integrating baroreceptor and chemoreceptor afferents (28). The cardiovascular responses of NMDA and non-NMDA receptors in the NTS have been reported (49). Hence, it is most likely that glutamatergic projections from vlPAG to the ionotropic receptors present in the NTS have a role in the HR regulation via baroreflex.

In the rest of our experiment, we evaluate the role of NMDA and non-NMDA receptors of vlPAG during Hem. In the Hem condition, Glu reversed hypotension induced by Hem and enhanced HR. Moreover, blockade of the Glu receptors through MK decreased the cardiovascular responses induced by Hem. Thus, it shows the role of the NMDA receptor in mediating the hemodynamic responses during Hem.

The role of NMDA and non-NMDA receptors in cardiovascular regulation during Hem condition is indicated (50). For the first time, we evaluated Hem-induced hemodynamic responses via ionotropic Glu receptors of vlPAG.

There is comprised of the effect of the glutamatergic system in Hem (51). Research points out that NMDA receptors are involved in Hem (52). It is assumed that the effect of Glu might be accompanied by other neurotransmitters such as glycine, norepinephrine, serotonin, acetylcholine, and GABA (53). These neurotransmitters alter the sympathetic activity during Hem in cardiovascular regions at the medulla level, including RVLM, NTS, and CVLM (53).

In line with the current study, another study revealed that mu receptor agonist microinjection into vlPAG reversed hemodynamic reflexes, followed by Hem (54). According to vlPAG and RVLM connections, it seems that the Glu pathway from vlPAG to RVLM had an excitatory effect on the cardiovascular reflexes induced by Hem. It is well documented that there is a GABAergic-Gluergic neural circuit in vlPAG, and activation or inhibition of each neural group can affect Glu neurons projecting to RVLM (43).

The serotonergic neurons of raphe nuclei are involved in the cardiovascular reflexes following Hem (55). Also, 5-HT1A receptors in RVLM participate in sympathoinhibitory responses during Hem (56). As mentioned earlier, there is a correlation between vlPAG and midline raphe nuclei (47). In addition, serotonergic neurons are one of the different neurons population in vlPAG (39). Thus, Glu’s possible mechanism attenuating hypotensive responses in Hem is that activated serotonergic neurons of vlPAG inhibit serotonergic neurons of raphe nuclei and cause disinhibition in RVLM. The raphe nuclei affect BP and HR via innervating preganglionic sympathetic neurons in the spinal cord, directly or via indirect projections to the CVLM or RVLM (54). Hence, it is possible that the Glu receptors participated in cardiovascular regulation during Hem through interaction with serotonin receptors of raphe nuclei.

VlPAG also projects arising neurons to the PVN (57). Also, PVN is involved in response to blood volume reduction via vasopressin secretion (58); therefore, it is possible that activating the Glu receptors in vlPAG via increase of vasopressin release from PVN also enhanced the cardiovascular parameters. Although, this hypothesis requires future experiments to prove it.

In the current study, the tachycardia due to blood withdrawal, enhanced by Glu microinjection. The mechanism of this impact is poorly documented. Though, baroreflex activates following Hem and maintains BP and HR close to the baseline. The presence of both NMDA and non-NMDA receptors has been indicated in the NTS that take part in the baroreflex (59, 60). Since the NMDA receptor antagonist in vlPAG attenuated HR, the mentioned receptor likely participates in HR regulation via NTS.

Conclusion

The present study revealed that activation of NMDA receptors of vlPAG enhanced the cardiovascular responses in normotensive and hemorrhagic hypotensive rats. Considering the inhibitory role of vlPAG, it seems the Glu does not have an important role in normotensive and hemorrhagic conditions. Concerning this neurotransmitter nature, it has a stimulatory effect.

Acknowledgment

We would like to thank the Research Council of Mashhad University of Medical Sciences, Mashhad, Iran for their financial support.

Funding Information

The results presented in this paper were part of a Ph.D. student thesis that was supported by research grants from the Research Council of Mashhad University of Medical Science.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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