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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 5 | Page : 326-334 |
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A glutamatergic pathway between the medial habenula and the rostral ventrolateral medulla may regulate cardiovascular function in a rat model of post-traumatic stress disorder
Ya-Yang Wu1, Cheng-Hong Zeng1, Kun-Yi Cai1, Chao Zheng2, Meng-Ya Wang3, Huan-Huan Zhang4
1 Psychophysiology Laboratory, Wannan Medical College; Cell Electrophysiology Laboratory, Wannan Medical College, Wuhu, Anhui, China
2 Neurobiology Laboratory, Wannan Medical College, Wuhu, Anhui, China
3 Cell Electrophysiology Laboratory, Wannan Medical College, Wuhu, Anhui, China
4 Psychophysiology Laboratory, Wannan Medical College, Wuhu, Anhui, China
| Date of Submission | 12-Jan-2023 |
| Date of Decision | 02-Apr-2023 |
| Date of Acceptance | 19-Apr-2023 |
| Date of Web Publication | 06-Jul-2023 |
Correspondence Address:
Prof. Huan-Huan Zhang
Wannan Medical College, Wuhu, Anhui 241002
China
Prof. Meng-Ya Wang
Wannan Medical College, Wuhu, Anhui 241002
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-23-00003
Post-traumatic stress disorder (PTSD) is a serious psychiatric disorder, and there is an association between it and the development of cardiovascular disease. The aim of this study was to explore whether there is a glutamatergic pathway connecting the medial habenula (MHb) with the rostral ventrolateral medulla (RVLM) that is involved in the regulation of cardiovascular function in a rat model of PTSD. Vesicular glutamate transporter 2 (VGLUT2)-positive neurons in the MHb region were retrogradely labeled with FluoroGold (FG) by the double-labeling technique of VGLUT2 immunofluorescence and FG retrograde tracing. Rats belonging to the PTSD model group were microinjected with artificial cerebrospinal fluid (ACSF) or kynurenic acid (KYN; a nonselective glutamate receptor blocker) into their RVLM. Subsequently, with electrical stimulation of MHb, the discharge frequency of the RVLM neurons, heart rate, and blood pressure were found to be significantly increased after microinjection of ACSF using an in vivo multichannel synchronous recording technology; however, this effect was inhibited by injection of KYN. The expression of N-methyl-D-aspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits was significantly increased in RVLM of PTSD model rats analyzed by the Western blotting technique. These findings suggest that there may be a glutamatergic pathway connection between MHb and RVLM and that this pathway may be involved in the regulation of cardiovascular function in the PTSD model rats, by acting on NMDA and AMPA receptors in the RVLM.
Keywords: Cardiovascular function, glutamate, medial habenula, post-traumatic stress disorder, rostral ventrolateral medulla
How to cite this article:
Wu YY, Zeng CH, Cai KY, Zheng C, Wang MY, Zhang HH. A glutamatergic pathway between the medial habenula and the rostral ventrolateral medulla may regulate cardiovascular function in a rat model of post-traumatic stress disorder. Chin J Physiol 2023;66:326-34 |
How to cite this URL:
Wu YY, Zeng CH, Cai KY, Zheng C, Wang MY, Zhang HH. A glutamatergic pathway between the medial habenula and the rostral ventrolateral medulla may regulate cardiovascular function in a rat model of post-traumatic stress disorder. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:326-34. Available from: https://www.cjphysiology.org/text.asp?2023/66/5/326/380731 |
| Introduction | |  |
Post-traumatic stress disorder (PTSD) refers to the delayed appearance and persistence of individual mental disorders caused by life-threatening events or severe trauma. Experiencing traumatic events, such as domestic violence, natural or man-inflicted disasters, serious traffic accidents, or military combat, can lead to the development of PTSD.[1] There is now growing evidence of a link between PTSD and the development of cardiovascular disease (CVD),[2] as several studies have shown that PTSD patients were often present with cardiovascular symptoms such as increased heart rate, elevated blood pressure, and arrhythmias.[3]
The medulla oblongata is the most basic center for regulating cardiovascular activities, including the depressor areas of the caudal ventrolateral medulla and the pressor area of the rostral ventrolateral medulla (RVLM). RVLM plays a key role in maintaining blood pressure, sympathetic cardiovascular tension, and the arterial baroreceptor reflex (ABR).[4],[5] RVLM contains a variety of amino acid transmitters and their corresponding receptors, that play an important role in the regulation of cardiovascular functions.[6] It has been reported that the administration of exogenous excitatory amino acids, such as aspartic acid and glutamate (Glu) to RVLM, can increase blood pressure.[7],[8] Glu in RVLM is an important excitatory neurotransmitter, as it can activate the corresponding Glu receptors to inflict changes in sympathetic cardiovascular activities, and Glu may be an important neurotransmitter for the ABR.[9]
The habenula (Hb) nucleus is an evolutionarily conserved ancient nucleus that exists in the brain of all vertebrates. It is divided into the lateral Hb and the medial habenula (MHb). Increased blood pressure caused by Hb neuronal excitation belongs to a defense response. Its involvement in cardiovascular regulation is similar to that of the hypothalamus. Hb is a necessary link that realizes the descending pathway of cardiovascular regulation,[10] and the neurons distributed in Hb are mainly glutamatergic. In MHb, the upper region contains glutamatergic neurons, the middle dorsal region contains substance P and glutamatergic neurons, and the lower, middle ventral, and lateral regions contain cholinergic and glutamatergic neurons.[11] The main symptoms of PTSD patients are long-term fear memory, avoidance, and increased alertness; notably, MHb is closely related to fear memory in many aspects.[12] Previous studies in our laboratory have suggested that an MHb excitability is increased in the rat PTSD model,[13] and it is speculated that the MHb may be involved in the regulation of cardiovascular function through the secretion of Glu.
Glu is an excitatory amino acid neurotransmitter that is mainly distributed within the central nervous system. In fact, a large number of synapses use Glu as a transmitter for neuromodulation. Glu receptors are divided into two categories: the ionic Glu receptors and the metabolic Glu receptors. In the central nervous system, the excitatory transmission between neurons is mainly mediated by N-methyl-D-aspartic acid (NMDA) receptors and by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.[14] NMDA and AMPA receptors in the RVLM are known to play important roles in controlling cardiovascular function.[15] Therefore, it is not clear whether the changes in the activity of the RVLM neurons in the rat model of PTSD are related to the secretion of the MHb-derived Glu, and if so, whether the secreted Glu regulates the cardiovascular function of the PTSD model rats by acting on NMDA and AMPA receptors in their RVLM.
In this study, we explored the mechanism underlying the potential connection between the MHb and the RVLM, and whether this connection is involved in the regulation of the cardiovascular function in PTSD model rats. To do so, we employed a double-labeling technology combining FluoroGold (FG) retrograde tracing and immunofluorescence histochemical staining, an in vivo multichannel synchronous recording technology, as well as Western blotting.
| Materials and Methods | | |
Experimental animals and groups
Adult male Sprague-Dawley rats (weighing 180–220 g) were provided by the Hangzhou Medical College (Hangzhou, Zhejiang, China). All experimental procedures were approved by the Experimental Animal Ethics Committee of the Wannan Medical College (LLSC-2022-247). The rats were randomly divided into the control and the PTSD model group. The rats were adaptively fed for a week; during this process, their eating activity (such as food intake and water intake), their daily activities, and their mental state were observed on a daily basis.
The PTSD model is based on the internationally recognized single-prolonged stress (SPS) approach that employs a compound stress (CS) approach in which the electrical stimulation (ES) is combined with SPS.[16] First, the rats were imprisoned for 2 h, and then, the plantar of the rats was subjected to an inescapable ES (30-V) for 2–10 sec, with random change intervals of 30–120 s, and a stimulation duration of 1 h. Subsequently, the rats were taken out for forced swimming (water temperature was about 20°C) that lasted until the rats gave up struggling and began to sink to the bottom due to exhaustion; at that point, the rats were removed from the bucket. Over the next 3 days, the rats were subjected to unavoidable electric plantar stimulation (30-V) for 10 min each day and were then placed in ventilated single cages. The control group was given the same feeding environment, without any special treatment. Behavioral tests were performed on the 7th day after the modeling began.
Behavioral testing
The weight of the rats was measured using an electronic scale before the modeling as well as before the undertaking of the behavioral experiments, and the weight gain of the rats was calculated by subtracting the previous weight from the last weight.
Open-field (OF) experiment: detection indicators included the total distance, the distance in the central region, and the stopover time in the central region. In the experiment OF, rats were put into the center OF for testing. The test area and parameters were set in advance in the Smart 3.0 system (Panlab, Harvard Apparatus, USA), and the exploration time was set at 5 min. After each trial, the field was cleaned and wiped with 75% alcohol to eliminate the odor left behind by the rats.
The elevated plus maze (EPM) test: the detection indexes included the ratio of the open arm entry (OE%) and the ratio of open-arm time (OT%). In the Smart 3.0 system, the open arm, the closed arm, and the central region were selected in advance, and then the parameters were set. After setting, each rat was placed in the central region toward the open arm, and its movement trajectory was recorded for 5 min. After each trial, the field was cleaned with 75% alcohol to eliminate the odor left behind by the rats.
FluoroGold retrograde tracing and immunofluorescence colocation
Perfusion, fixation, and sectioning
Rats in the control group were given an intraperitoneal injection of mixed anesthetics (14 g urethane, 0.7 g chloralose, and 0.7 g borax per 100 ml normal saline, 0.6–0.7 mL/100 g), and were fixed after anesthesia. After referring to a previous study of ours,[13] fluorochrome FG (0.5 μL; 4%; Fluorochrome Inc., Denver, USA) was slowly injected into the RVLM for 5 min through the syringe pump (KDS 130; KD Scientific, USA) and remained for 10 min after the injection. Subsequently, the microinjection needle was slowly drawn out. After the rats were placed in ventilated single cages for 7 days, the rats that were injected with FG were anesthetized, needles were inserted into their left ventricles, and the rats were injected with normal saline and 4% paraformaldehyde. After the completion of this process, the brain tissue was put into 4% paraformaldehyde overnight, and then, the rat brain tissue was removed and successively dehydrated in 20% and 30% sucrose until it sank to the bottom, after which it was removed and embedded. Brain tissue sections of 30 μm were subsequently prepared in a cryostat box microtome (HM525 NX, Thermo Scientific, Germany).
Immunofluorescence reaction
Sections were drilled with 0.3% Triton X-100 for 2 h, were sealed with goat serum for 2 h, and were incubated overnight at 4°C with a rabbit anti-vesicular Glu transporter 2 (VGLUT2) (1:200; DF13296; Affinity Biosciences, China) primary antibody. The sections were then incubated with a goat anti-rabbit IgG (H+L) (1:200; BA1032; Boster Biotechnology Inc., China) secondary antibody were incubated for 2 h, and the slices were sealed. After each step, the brain sections were rinsed thoroughly with 0.01 M PBS (pH 7.3), and the results were observed under an inverted fluorescence microscope (IX3; Olympus, Japan).
Multichannel simultaneous recording in vivo
Based on a previous study of ours,[13] the rats were anesthetized and intubated in the right femoral artery, and the blood pressure sensor was connected to the bridge amplifier (ML 221, ADInstruments, Australia). A standard limb II lead electrocardiogram was collected with a needle electrode. Subsequently, the rats were placed in the double-arm stereotaxic apparatus (Model 68002, Shenzhen Ruiwo De Life Technology Co., Ltd., China) and were fixed. The right RVLM (12.00–12.36 mm posterior to the bregma, 1.90–2.40 mm lateral to the midline, and 8.10–8.50 mm below the meninges) and the right MHb (2.52–4.20 mm posterior to the bregma, 0.15–0.60 mm lateral to the midline, and 3.8–4.6 mm below the meninges) were located using the Paxinos and Watson brain atlas.[17] The rat skull was drilled in the corresponding position, and the self-made drug delivery and recording electrode (electrode resistance: 5-15 MΩ) were lowered into the RVLM, and the concentric stimulation electrodes (KD-CS; Kedou BC, China) were lowered into the MHb. The PTSD model group rats were divided into two groups: those receiving artificial cerebrospinal fluid (ACSF; hereafter, the “ACSF group”) and those receiving kynurenic acid (KYN; hereafter, the “KYN group”). After the electrodes were placed in the nuclei, the PowerLab system collected evidence of stable spontaneous discharges by the RVLM neurons for 20 min using an amplifier (A-M Systems 1800), and then 0.2 μL of ACSF or 0.2 μL of KYN (10 nmol/200 nL; K3375; Sigma, USA) was injected.[18] After 10 min, the MHb was electrically stimulated (200 and 400 μA, 0.2 msec, 100 Hz, 0.2 s) with a stimulator (Model 2100; A-M Systems, USA) to observe the effects of the ES on the neuronal discharge signals of RVLM, the blood pressure, and the heart rate. The above-observed parameters were obtained by LabChart software (ADInstruments, Australia).
Western blotting analysis
The rats were anesthetized and sacrificed; then, their brains were removed and placed in a refrigerator at –80°C. According to the brain atlas, the RVLM tissue was perforated in coronal sections of the brain stem.[19] The protein concentration was determined by the bicinchoninic acid method. The protein samples were then placed into 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were then transferred onto a polyvinylidene fluoride membrane, were blocked with 5% skim milk for 2 h, and the membranes were incubated overnight at 4°C with rabbit anti-AMPA receptor (GluR2) (1:1,000; A11316; ABclonal Technology Co., Ltd., China), rabbit anti-NMDAR1 (1:1,000; A11699; ABclonal Technology Co., Ltd., China), and rabbit anti-GAPDH (1:20,000; AF7021; Affinity Biosciences, China) primary antibodies. The membranes were then placed in goat anti-rabbit IgG-HRP (1:10,000; BA1054; Boster Biotechnology Inc., China) secondary antibody and incubated for about 2 h. The gray value of the protein bands was measured, and the gray value ratio of the target protein bands to the GAPDH bands was used to reflect the expression level of each target protein.
Statistical analysis
Data were expressed as mean ± standard error of the mean. An independent samples t-test was used for the undertaking of comparisons between different groups, while one-way repeated measures ANOVA was used for comparisons among levels recorded before and after stimulation within the same group. SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used for the undertaking of the statistical analysis, and P < 0.05 was considered statistically significant.
| Results | | |
Identification of post-traumatic stress disorder model rats
Body weight gain
After 7 days of modeling, the weight gain was significantly lower in the PTSD model group than in the control group (P < 0.01) [Figure 1]. | Figure 1: Analysis of the weight change data of the control (n = 12) and the PTSD model (n = 12) groups after 1 week of modeling. Data represent the mean ± SEM, and comparisons between groups were made by the independent samples t-test. **P < 0.01. PTSD: Post-traumatic stress disorder, SEM: Standard error of the mean.
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Behavioral test
In the OF test, the total distance and the distance in the central region were found to be significantly reduced in the PTSD model group compared to the control group (P < 0.01) [Figure 2], and so was the stopover time in the central region (P < 0.05) [Figure 2]. | Figure 2: Representative tracking plots of the OF test for each group at the end of modeling (a and b) and comparative data plots of the relative changes of different experimental parameters between the groups (c-e). Data represent the mean ± SEM. The control (n = 12) and the PTSD model (n = 12) groups were compared using the independent samples t-test. *P < 0.05, **P < 0.01. PTSD: Post-traumatic stress disorder, SEM: Standard error of the mean.
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In the EPM test, both the OE% and the OT% of the PTSD model group were found to be significantly lower than those of the control group (P < 0.01) [Figure 3]. | Figure 3: Representative tracking plots of the EPM test (a and b) and comparative graphs of the changes in OE% and OT% data for each group (c and d). Data represent the mean ± SEM. Both the control (n = 12) and the PTSD model (n = 12) groups were compared using the independent samples t-test. **P < 0.01. PTSD: Post-traumatic stress disorder, SEM: Standard error of the mean.
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Glutamatergic pathway linkage between MHb and RVLM
Morphological observation of the glutamatergic pathway linkage between MHb and RVLM
FG is a retrograde tracer that is frequently used to study the presence of neural pathway connections between neurons. FG was microinjected into the RVLM, and brain tissue sections were placed under an inverted fluorescence microscope to observe the distribution of neurons. Retrogradely labeled neurons were detected in the MHb [Figure 4]a and [Figure 4]d. The neuronal cell bodies and fibers were found to be golden-yellow in color. VGLUT2 immunofluorescence revealed the VGLUT2-positive neurons in the MHb region, with orange-colored cytosol and fibers [Figure 4]b. Golden and orange double-labeled neurons were colored orange-red [Figure 4]c. [Figure 4]e shows the negative control of [Figure 4]b, [Figure 4]f is a merged view of [Figure 4]d and [Figure 4]e. While [Figure 4]i reveals the injection site in the RVLM. In addition, as it is known that downstream fibers from the hypothalamic paraventricular nucleus (PVN) neurons can project directly into the RVLM, we observed that the cells in the PVN region [Figure 4]g and [Figure 4]h were also retrogradely labeled by FG. | Figure 4: FG-mediated retrograde tracing with immunofluorescence colocalization and an injection site map, after the microinjection of FG into the RVLM. (a and d) FG-mediated retrograde labeling of the MHb. (b) The area indicated by the yellow arrow is the VGLUT2-positive area of the MHb. (c) Composite image of (a and b). (e) The yellow arrow indicates the MHb regions' VGLUT2-negative control. (f) Composite image of (d and e). (g and h) Yellow arrows point to the area of the retrograde marking of PVN. (i) The yellow arrow indicates the RVLM injection site. Scale bars: (a − c), 50 μm; (d − f) and (h), 100 μm; (g) and (i), 200 μm. FG: FluoroGold, RVLM: Rostral ventrolateral medulla, MHb: Medial habenula, PVN: Paraventricular nucleus, VGLUT2: Vesicular glutamate transporter 2.
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Functional investigation of the glutamatergic linkage between MHb and RVLM
PTSD model rats were electrically stimulated in their MHb areas after ACSF or KYN were administered to their RVLM, and the changes in their RVLM neuronal discharge frequency and their cardiovascular activity were observed.
As a result, it was observed that the firing frequency of the RVLM neurons in the ACSF group was significantly higher when a 400-μA ES was applied to the MHb (P < 0.05) [Figure 5]a and [Figure 5]b. In contrast, the discharge frequency of the RVLM neurons in the KYN group was found to be significantly decreased when a 200-μA ES was applied (P < 0.05) [Figure 5]c and [Figure 5]d. | Figure 5: Raw graphs of the changes in the RVLM neuronal discharge frequency before and after an ES in the ACSF and the KYN groups (a and c), and comparison of the discharge frequency data before and after the stimulation (b and d), with the arrows pointing to the ES points. (b): At 200 μA, n = 9; at 400 μA, n = 9. (d): At 200 μA, n = 9; at 400 μA, n = 9. Data represent the mean ± SEM. One-way repeated measures ANOVA was used for the comparison of the levels before and after the ES. *P < 0.05 versus before ES. RVLM: Rostral ventrolateral medulla, ACSF: Artificial cerebrospinal fluid, ES: Electrical stimulation, SEM: Standard error of the mean.
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After the administration of a 400-μA ES to the MHb, the heart rate in the ACSF group was found to be significantly accelerated (P < 0.05) [Figure 6]a and [Figure 6]b; however, the heart rate of rats in the KYN group decreased significantly after 200-μA ES (P < 0.05) [Figure 6]c and [Figure 6]d. | Figure 6: Histograms of the heart rate changes observed before and after the ES in the ACSF and the KYN groups (a and c), and comparison of the heart rate data before and after the ES (b and d), with arrows pointing to the stimulation points. (b and d): At 200 μA, n = 9; at 400 μA, n = 9. Data represent the mean ± SEM. One-way repeated measures ANOVA was used for the comparison of the levels before and after the ES. *P < 0.05 versus before ES. ACSF: Artificial cerebrospinal fluid, ES: Electrical stimulation, SEM: Standard error of the mean.
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After the administration of a 400-μA ES in the MHb, there was no significant difference identified in the rats' systolic blood pressure (SBP) and the diastolic blood pressure (DBP) before and after the stimulation in the ACSF group; however, the mean arterial pressure (MAP) were found to be significantly increased (P < 0.05) [Table 1] and [Figure 7]a. After a 200-μA ES, the rat SBP (P < 0.05) [Table 2] and [Figure 7]b was found to be significantly lower in the KYN group, while there was no statistically significant difference in terms of the recorded rat DBP and MAP [Table 2]. | Figure 7: The original graphs of the ABP before and after an ES were delivered in the ACSF (a) and the KYN (b) groups, with the arrows pointing to the ES points. ABP: Arterial blood pressure, ES: Electrical stimulation.
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 | Table 1: Analysis of the rat blood pressure changes before and after the delivery of ES to the artificial cerebrospinal fluid group
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 | Table 2: Analysis of the rat blood pressure changes before and after the delivery of ES to the kynurenic acid group
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Changes in the RVLM content of glutamate receptors
When compared with the control group, the protein expression of the NMDA receptor subunit NMDAR1 and the AMPA receptor subunit GluR2 in the RVLM of the PTSD model group revealed a more significant trend of increase (P < 0.05) [Figure 8]. | Figure 8: Original Western blot plots of the control and the PTSD model groups (a), and comparative plots of data on the relative protein expression changes (b and c). Data represent the mean ± SEM. An independent samples t-test was used to compare the control group (n = 7) with the PTSD model group (n = 7). *P < 0.05. PTSD: Post-traumatic stress disorder, SEM: Standard error of the mean.
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MHb and RVLM sites' identification
At the end of the experiment, brain tissue was taken and fixed in 4% paraformaldehyde, coronal sections were produced, and the corresponding needle tract sites were compared to the coordinates of the Paxinos and Watson brain atlas so as to determine the accuracy of the recording and the ES sites [Figure 9]. | Figure 9: Map of the ES and the recording sites. (a) MHb ES location. (c) RVLM recording location. (b and d) Corresponding MHb and RVLM coronal maps obtained from the brain atlas. MHb: Medial habenula, RVLM: Rostral ventrolateral medulla, ES: Electrical stimulation.
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| Discussion | | |
When faced with natural disasters, war, terrorist attacks or threats to life, and psychological emotions such as anxiety and irritability may lead to the development of PTSD. A growing number of studies are now finding that PTSD also affects cardiovascular function. The present study investigates the central regulatory mechanisms of the MHb-RVLM pathway on cardiovascular function in PTSD.
Retrograde tracing is a common tracing method used in neuroscientific research to trace the origin of certain nuclei afferent nerves in the nervous system, by combining the immunofluorescence methods so as to qualitative analysis of the pathway. By microinjecting the retrograde tracer FG into the RVLM region, we observed retrogradely labeled neurons in the MHb and the PVN regions. Retrograde labeling and VGLUT2-positive double-labeled cells were observed in the MHb region. It has been reported that PVN has a downward fiber projection to the RVLM,[20] and retrogradely labeled neurons were observed in the PVN region, thereby further validating the accuracy of the injection site. Glutamatergic neurons release Glu that they transport through vesicular Glu transporters (VGLUTs), which are divided into three categories: VGLUT1, VGLUT2, and VGLUT3. VGLUT1 and VGLUT2 mRNAs are widely distributed across the glutamatergic neurons of the brain, while VGLUT3 mRNA has been found to be expressed in some non-glutamatergic neurons.[21],[22] Among them, VGLUT1 and VGLUT2 are highly specific markers of glutamatergic neurons and of their axon terminals that can better label glutamatergic nerve endings and glutamatergic synapses.[23] Since VGLUT1 is mainly distributed in the cortex, the hippocampus, and the cerebellar cortex, VGLUT2 can be found in the thalamus, spinal cord, and medulla oblongata.[24],[25] In our study, VGLUT2 was used as a glutamatergic neuronal marker based on the location of the VGLUT2 distribution. Based on our findings, it was suggested that there may be downstream glutamatergic neuronal projections from the MHb to the RVLM.
Based on the experience gained by a previous study undertaken in our laboratory,[13] we, herein, used the CS composite method to establish the PTSD rat model, and we verified the successful modeling through the weight gain change, and the OF and EPM experiments' results, as well as on the fact that the rats in the PTSD model group exhibited significant anxiety and other emotions. One study has found that PTSD contributes to CVD.[3] Previous laboratory studies have shown that the excitability of MHb and RVLM regions in PTSD model rats was enhanced. When different intensities of ES were given to MHb, RVLM discharge frequency was increased, and cardiovascular activities were enhanced, which indirectly indicates that there may be a certain functional connection between MHb and RVLM, and it may be involved in the regulation of cardiovascular activities.[13] However, the exact mechanism of this regulation is unclear. In the current study, we observed that the RVLM neuronal discharge frequency, the heart rate, and the MAP in the ACSF group increased to different degrees when the MHb was given an ES of different intensities, and these effects were inhibited and produced a downward trend by the injection of the nonselective Glu receptor blocker KYN. The RVLM is the main source of cardiac and vascular sympathetic excitatory activity, and the excitation of this region leads to increased sympathetic output, thereby resulting in increased blood pressure.[4] A study has found that the microinjection of KYN in the RVLM of some rats in a hypertension model was able to inhibit the sympathetic excitation; however, no significant response was recorded after the same injection was administered to control rats, thereby suggesting that this may be related to the increased number or sensitivity of Glu receptors in the RVLM.[25],[26] Therefore, the inhibitory effect is more significant when KYN is injected. The above results suggest that in the PTSD model rats, the MHb may increase the sympathetic excitability by secreting Glu to act on the RVLM, thereby increasing both the blood pressure and the heart rate of these rats.
We also observed that the NMDA and AMPA Glu receptors in the RVLM are involved in sympathetic excitability regulation in the PTSD model rats. Studies have reported that the risk of hypertension and CVD increases with the exacerbation of PTSD, which may be associated with alterations in the autonomic nervous system, and might possibly refer to a decreased excitability of the resting parasympathetic nervous system, an increased excitability of the sympathetic nervous system,[27],[28],[29],[30] and a dysregulation of the hypothalamic-pituitary-adrenal axis.[31] The RVLM is an important center for the regulation of sympathetic and cardiovascular activity.[32] The NMDAR1 subunit is essential for NMDA receptor function, which is the basic subunit of the ion channel; hence, the NMDAR1 subunit is usually used as a general marker for NMDA receptors.[33] AMPA receptor is mainly composed of GluR1, GluR2, GluR3, and GluR4 four subunits, of which the GluR2 subunit plays a key role in the biophysical properties of the AMPA receptor; the level of this content determines the function of AMPA receptor.[34] Therefore, NMDAR1 and GluR2 subunits were used as NMDA and AMPA receptor markers in this experiment. The Western blotting analysis revealed an increased expression of the NMDA and the AMPA receptors within the RVLM region in the PTSD model group. Based on previous experimental results, we hypothesized that the increased NMDA and AMPA receptors in the PTSD model group might be related to an increased release of Glu from the MHb. Of course, the alteration of the cardiovascular function in PTSD involves a comprehensive regulation of multiple neurotransmitters and other factors, and this experimental study only explores this mechanism through one aspect of it, while the research undertaken focuses only on the protein receptor level. As a result, the specific signaling pathway mechanism needs to be further explored in future studies.
| Conclusion | | |
In summary, the MHb may have a downstream glutamatergic projection to the RVLM, and the increased excitability of the MHb in PTSD model rats as well as the release of Glu to the RVLM, may be involved in the regulation of the cardiovascular activity in PTSD. This pathway seems to act through the NMDA and the AMPA Glu receptors of the RVLM, thereby providing an effective therapeutic target for the development of future approaches to the clinical treatment of CVD caused by PTSD.
Acknowledgments
The authors sincerely thank Xin Gong for the technical assistance.
Author contributions
Ya-Yang Wu: Conceptualization, methodology, validation, investigation, formal analysis, and writing the first draft. Cheng-Hong Zeng: Investigation. Kun-Yi Cai: Raising rats. Chao Zheng: Writing review and editing and funding acquisition. Meng-Ya Wang: Supervision, funding acquisition, and resources. Huan-Huan Zhang: Visualization, funding acquisition, paper review and revision, and supervision.
Financial support and sponsorship
This work was supported by the Natural Science Research Project of Colleges and Universities in Anhui Province (KJ2018A0266), the National Natural Science Foundation of China (31271155, 31200828), the Natural Science Foundation of Anhui Province, China (2108085MC91), and the Academic Funding Project for Excellent and Top-notch Talents in Colleges and Universities in Anhui Province, China (gxbjZD2021061).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2]
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