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. 2021 May 5;42(7):2205–2217. doi: 10.1007/s10571-021-01094-3

Upregulation of Glutamatergic Receptors in Hippocampus and Locomotor Hyperactivity in Aged Spontaneous Hypertensive Rat

Patrick Szu-Ying Yen 1,#, Yen-Chin Liu 2,#, Chun-Hsien Chu 3, Shiou-Lan Chen 1,4,✉
PMCID: PMC11421636  PMID: 33954807

Abstract

Epidemiologic studies have indicated that chronic hypertension may facilitate the progression of abnormal behavior, such as emotional irritability, hyperactivity, and attention impairment. However, the mechanism of how chronic hypertension affects the brain and neuronal function remains unclear. In this study, 58-week-old male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) control rats were used. Their locomotor activity and neuronal function were assessed by the open field test, novel object, and Y maze recognition test. Moreover brain tissues were analyzed. We found that the aged SHR exhibited significant locomotor hyperactivity when compared to the WKY rats. However, there was no significant difference in novel object and novel arm recognition between aged SHR and the WKY rats. In the analysis of synaptic membrane protein, the expression of glutamatergic receptors, such as the N-methyl-D-aspartate (NMDA) receptor receptors subunits 2B (GluN2B) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 1 (GluA1) in the hippocampus of SHR were significantly higher than those of WKY rats. In addition, in the synaptic membrane of SHR's hippocampus and medial prefrontal cortex (mPFC), a down-regulation of astrocytes was found, though the excitatory amino acid transporter 2 (EAAT2) remained constant. Moreover, a down-regulation of microglia in the hippocampus and mPFC was seen in the SHR brain. Long-term exposure to high blood pressure causes upregulation of glutamate receptors. The upregulation of glutamatergic receptors in hippocampus may contribute to the hyper-locomotor activity of aged rodents and may as a therapeutic target in hypertension-induced irritability and hyperactivity.

Keywords: Aged rat, Glutamatergic receptors, Hypertension, Locomotor hyperactivity

Introduction

Currently, hypertension is an established risk factor for cerebrovascular disease (Kario et al. 2002; Tocci et al. 2017); with more and more evidences linking hypertension with the changes of neuronal function (Nishtala et al. 2015; Walker et al. 2017; Foulquier et al. 2018). In animal studies, the spontaneously hypertensive rats (SHR) were originally developed as a model for chronic hypertension but later on showed accompanying pathologies such as attention-deficit and hyperactivity disorder (ADHD) and cognitive decline (Grunblatt et al. 2015; Sagvolden and Johansen 2012). In many studies, SHR was used as a model for ADHD (Tsai and Lin 1988; Fasmer and Johansen 2016; Sagvolden 2000; Kishikawa et al. 2014). Since the key characteristics of ADHD patients include increase of locomotor activity (Teicher 1995; Dekkers et al. 2020), attention-deficit (Arns et al. 2009), and impulsive behavior (Avila et al. 2004; Brook and Boaz 2005), this increase of locomotor activity in SHR (Tsai and Lin 1988; Fasmer and Johansen 2016) coincides with the defining characteristic of patients with ADHD. However, the mechanism that induces SHR locomotor hyperactivity is not well known.

Studies have shown that the activation of glutamatergic system in hippocampus (Zhang et al. 2002) and medial prefrontal cortex (mPFC) (Lacroix et al. 1998; Kehr et al. 2018) are involved in increasing locomotor activity in rodents. In various animal models of hypertension, the increase in glutamatergic excitatory input to pre-sympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus was believed to lead to an increase in sympathetic nerve outflow (Tsuda 2013; Li and Pan 2017) and was considered to be the main cause of hypertension (Mancia and Grassi 2014). However, the expression of glutamatergic receptors in hippocampus and mPFC in chronic hypertension is currently unknown. The roles of glutamatergic receptors in SHR hyperactivity needed to be elucidated. Evaluating the level of glutamatergic receptors in the brain of SHR can help us clarify the mechanism of hyper-locomotor activity in chronic hypertension and may provide relevant references for pharmacological treatment.

Moreover, studies have revealed that SHR develops neuronal inflammation and affects their ability to modulate blood pressure in PVN (Masson et al. 2015; de Almeida Silva et al. 2020). Microglia, the resident immune cell cells in the brain, is what initiates and modulates neuroinflammation, which may, in turn, contribute to the elevated blood pressure (de Almeida Silva et al. 2020) and neuronal function (Kohman 2012). Although microglia in the hypothalamic PVN were found to be more activated in SHR than in the control rats (Masson et al. 2015; de Almeida Silva et al. 2020), a down-regulation of microglia cells in the rostral ventrolateral medulla of SHR was noted (Cohen et al. 2019). Therefore, the controversial roles of microglia in hypertension need to be elucidated. The degrees of neuronal inflammation in the hippocampus and mPFC in chronic hypertension need to be further explored. Evaluating the expression of microglial cells in the hippocampus and mPFC in SHR may help us clarify the relationship of neuronal inflammation with the hyper-locomotor activity in chronic hypertension.

To understand the changes of neuronal function and mechanism induced by chronic hypertension, 58-week-old SHR and Wistar-Kyoto (WKY) control rats were used. The locomotor activity and neuronal function of rats were tested by open field, novel object, and Y maze recognition test. To better grasp the mechanism and pathophysiological changes of brain in SHR, the expression of glutamatergic receptors, such as N-methyl-D-aspartate (NMDA) receptor receptors subunits 2A (GluN2A) and 2B (GluN2B), glutamate receptor 1 (GluA1) and receptor 3 (GluA3) in hippocampus and mPFC were evaluated. The expression of postsynaptic density protein 95 (PSD-95) were also been evaluated. Furthermore, the degrees of neuronal inflammation were checked by evaluating the expression of microglia and astrocyte in mPFC and hippocampus.

Methods

Animal

Studies were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) regulations, the US Department of Agriculture Animal Welfare Act, and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (KMU) and adhered to the ethical standards established by the IACUC ethics committee of the KMU.

Male 58-week-old spontaneously hypertensive rats (SHR) and its counterpart, the Wistar-Kyoto (WKY) rats, were purchased from the National Laboratory Animal Center in Taiwan and were kept in an environmentally controlled room in an AAALAC-certified breeding facility of KMU (temperature: 23 ± 2 °C; 12-h/12-h light/dark cycle with light on from 07:00 to 19:00) and had free access to food and water during the experiment.

Open Field Test

Animals were transported to the testing room and left undisturbed for 60 min before the test. Room brightness was kept consistent, and there were no disturbing sounds or odors during the tests. The same experimenter conducted all tests. A camera and image-tracking software (Panlab Smart 3.0 video-tracking software) were used to monitor and analyze rat behavior.

The open field test was used to test the locomotor activity in rat. Testing is performed in a black square acrylic box (80 × 80 × 40 cm) (Fig. 1) for 10 min. After each test, the maze was wiped with a 75% alcohol solution to prevent odor cues. The travel distance (cm) and traveling speed (cm/min) were used as indictors of locomotor activity.

Fig. 1.

Fig. 1

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The body weight and blood pressure in 58-week-old spontaneously hypertensive rats (SHR, n = 8) and its counterpart, the Wistar-Kyoto (WKY, n = 11) control rats. The body weight [gm] (a), diastolic blood pressure [DBP, mmHg] (b), and systolic blood pressure [SBP, mmHg] (c) are shown for the indicated group. Data are presented as the mean ± SEM. **p < 0.01, ***p < 0.001 vs WKY control rat (t-test)

Novel Object Recognition Test

We quantified cognitive function in rat by using the novel object recognition test, which is a behavioral test used to measure a rodent’s tendency to explore new object. Rodents with normal cognitive function typically prefer to investigate a new object rather than the familiar object (Fig. 2a). The cognition and short-term memory-related areas of the brain such as the mPFC and hippocampus are involved in this task. In brief, a rat was presented with two similar objects during the first session trial (T1), and then a new object replaces one of the two objects in T1during a testing trail (T2). Testing was performed in a black square acrylic box (80 × 80 × 40 cm) (Fig. 2a). During T1, rats were allowed to freely explore the two familiar objects for 10 min. After the T1 trial, the rat was returned to its cage for a 90-min inter-trial interval. In T2, the rat was placed into the medial area and then allowed to access all three areas (familiar object area [Zone 1], medial area, and novel object area [Zone 2]) of the box for 10 min. After each test and between T1 and T2, the maze was wiped with a 75% alcohol solution to prevent odor cues. The travel distance and the time spent in each area were recorded and analyzed.

Fig. 2.

Fig. 2

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The open field test data in 58-week-old spontaneously hypertensive rats (SHR, n = 8) and its counterpart, the Wistar-Kyoto (WKY, n = 11) control rats. The total travel distance [cm] (a), travel speed [cm/min] (b) and resting time [sec] (c) is shown for the indicated group. The moving track of WKY control rat (d) and SHR (e) is shown. p values are determined by using t-test

Y Maze Novel Arm Recognition Test

We also quantified the cognitive function in rat by using the Y maze test. Rodents with normal cognitive function typically prefer to investigate a new arm of the maze rather than return to the one previously visited (Maurice et al. 1994). Testing is performed in a maze with 3 black plastic arms: A, the start arm; B, the familiar arm; and C, the novel arm. The dimensions of each arm are as follows: 50 cm (length) × 10 cm (width) × 30 cm (height). These arms were placed at 120° angles to form a Y-shape with a medial area in the center (Fig. 3a).

Fig. 3.

Fig. 3

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The novel object recognition test data in 58-week-old spontaneously hypertensive rats (SHR, n = 8) and Wistar-Kyoto (WKY, n = 11) rats (a). The moving track of WKY control rats and SHR (b) at T2 is shown. The total travel distance [cm] (c) is shown for the indicated group. Data are presented as the mean ± SEM. **p < 0.01 SHR vs WKY (t test). The percentage [%] of travel distance in familiar [zone 1] and novel object [zone 2] zone (d) and % of travel time in in familiar and novel object zone (e) is shown for the indicated group. *p < 0.05, **p < 0.01 represent the significant difference in familiar and novel object zone within same group (t-test)

At the beginning of each test day, the rat was habituated to the testing room for 60 min. The test consisted of a 10-min first session trial (T1) and a 10-min testing trial (T2). In T1, the rat was placed at the end of arm A and allowed to freely explore arm A and B, with arm C blocked. After the T1, the rat was returned to its home cage for a 90-min inter-trial interval. In T2, the rat was placed at start arm A and then allowed to access all three arms of the maze (Fig. 3a). After each test and between T1 and T2, the maze was wiped with a 75% alcohol solution to prevent odor cues. The number of arm entries, the travel distance, and the time spent in each area were recorded and analyzed.

Invasive Blood Pressure Measurement

Before the euthanasia of rat, the intra-arterial blood pressure (BP) was measured with procedures as previously described (Shiou et al. 2018). Briefly, the catheterization was performed after the behavior tests. Rats were anesthetized with intraperitoneal pentobarbital sodium (60 mg/kg) and the femoral artery was cannulated. The cannula consisted of sterile polyethylene tubing with an internal diameter of 0.5 mm and an outer diameter of 0.9 mm provided with a 26 G × 1/2″ needle. Calibration was performed using a mercury sphygmomanometer. The whole setup was allowed to stabilize for 10 min before data collection on a data acquisition system (IX-RA-834, iWorx Systems, Dover, New Hampshire) with a pressure transducer.

Isolation of Synaptic Protein

After the behavioral tests, animals were euthanized with an overdose of pentobarbital. The brain was dissected and cut into two halves. The left hemispheric brains were frozen in liquid nitrogen immediately and used in Western blot analysis. The right hemi-brain was then separated into the areas related to the locomotor activity and cognitive function, such as the hippocampus and mPFC. Brain tissues were homogenized and lysed in synaptic protein extraction reagent (catalog number 87793, Thermo), supplemented with 1X protease inhibitor cocktail (catalog number 78441, Thermo). The homogenate was centrifuged at 4 °C for 10 min at 1000 × g. The supernatant was then collected and was further centrifuged at 15,000 × g for 20 min at 4 °C. The pellet, composed of the intact membranes and protein complexes of synapses, was re-suspended with the extraction buffer and stored in the -80 °C refrigerator until analysis.

Western Blotting

Equal amounts of synaptic protein (30 μg) were loaded and separated by 4–12% SDS-PAGE (SurePAGETM, Bis–Tris), then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% bovine serum albumin (BSA) in TBST for 1 h at room temperature and then incubated with primary antibodies for GluN2A (Cell Signaling, 4205, 1:1000), GluN2B (Cell Signaling, 4207, 1:1000), GluA1 (abcam, ab31232, 1:1000), GluA3 (abcam, ab40845, 1:2000), PSD-95 (Invitrogen, 51–6900, 1:500), astrocyte (anti-glial fibrillary acidic protein, GFAP, ab7260, 1:5000), EAAT2 (abcam, ab41621, 1:3000), and GADPH (Santa cruz, sc-32233, 1:2000) antibodies (dissolved in 5% BSA/TBST) for 12 h at 4 °C; and then incubated with secondary antibodies for 1 h at 25 °C. All of the blots were incubated and then visualized with ECL Western blot detection reagents (Thermo, 34,577).

Immunohistochemistry and Immunofluorescence Staining

The right half of brain was placed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4 °C. After the brain had been suspended in a sucrose solution (10–30%), it was embedded in optimal cutting temperature compound and frozen immediately at –80 °C. Serial transverse brain slices (30 μm) were collected from PFC and hippocampus by using a cryostat. Next, at least 6–7 brain slices were randomly selected from each brain area of rat. The randomized brain slice of mPFC and hippocampus might represent the expression of cells and markers in each brain area. To detect microglia, brain sections were soaked at 4 °C in 5% BSA mixed with PBST then incubated with anti-IbA1 (1:2000, WAKO-016–20,001) for 24 h at 4 °C. After the sections had been repeatedly washed in PBST, they were incubated in 1:1000 dilutions of biotinylated secondary antibody (BA-1000, Vector Laboratories, Peterborough, UK) and then in an avidin–biotin complex (Elite kit; Vector). The peroxidase reaction product was visualized by incubating the sections in a solution that contained 0.022% 3,30-diaminobenzidine (DAB) (Vector).

For immunofluorescence staining, brain slices in each brain area were incubated at 4°C in 5% BSA mixed with PBST and incubated with astrocyte marker glial fibrillary acidic protein (GFAP, abcam, ab7260, 1:1000), NeuN (Millipore, ABN78, 1:500), GluN2B (Abcam, ab65783, 1:100), and EAAT2 (abcam, ab41621, 1:2000) overnight at 4 °C. After washing with PBST, secondary antibody directed against rabbit IgG coupled to Alexa Fluor® 488 (Invitrogen A-21206, 1:200) and mouse IgG coupled to DyLight® 550 (Bethyl, A90-241D3, 1:200) for immunofluorescence detection were used. DAPI (4′,6-diamidino-2-phenylindole) was used as a nuclear counterstain. Finally, the sections were rinsed and mounted with Shandon Immu-Mount (Thermo Scientific) or Micromount solution (M-3801730, Leica Camera AG, Wetzlar, Germany).

The processed sections were examined with an upright microscope (BX53, Olympus, Tokyo, Japan) and an Olympus DP73 camera. For the acquisition of image, the same exposure time of brain area between groups were carefully controlled by the DP73 camera. The white or black balance were used to clear the noise of background before the image were captured. The OLYMPUS cellSens Dimension 1.13. software were used to analyze the expression of Iba1, GFAP, NeuN, and GluN2B in each brain area. In brief, each single plane image were turned into gray-scale before analysis. In order to evaluate the marker expression between groups of mice, the same calculation threshold was used for each markers and the intensity of immuno-signal of markers in unit area of brain slice were evaluated. The expression of markers were calculated by the positive cells counts or percentage (%) of immuno-intensity in unit of brain area compared to the WKY rat.

Statistical Analysis

Data were analyzed by using one-way analysis of variance and then Newman–Keuls post hoc test. Prism 5 and SPSS 22 were used to perform statistical analyses. Data are expressed as mean ± standard error of the mean. Significance was set at p < 0.05.

Results

Physiological Analysis

There were no significant difference in the body weight of SHR and WKY rats (SHR vs WKY rat: 440.5 ± 12.3 vs 418.4 ± 17.2 gm, p = 0.312, Fig. 1a). The SHR had significant higher diastolic pressure (DBP) (SHR vs WKY rat DBP: 163.6 ± 5.7 vs 94.5 ± 3.3 mmHg, p = 0.0001, Fig. 1b) and systolic blood pressure (SBP) (SHR vs WKY rat SBP: 244.0 ± 18.0 vs 143.8 ± 7.4 mmHg, p = 0.002, Fig. 1c) compared with the age-matched WKY control rats.

SHR Showed Significant Locomotor Hyperactivity Behavior than the WKY Rats

In open field behavior test, SHR exhibited significant locomotor hyperactivity compared to the WKY rats. SHR had higher travel distance (SHR vs WKY, 3483 vs 1914 cm, p < 0.0001, Fig. 2a), travel speed (SHR vs WKY, 5.9 vs 3.2 cm/min, p < 0.0001, Fig. 2b), and less resting time (SHR vs WKY, 147.4 vs 328.6 s, p < 0.0001, Fig. 2c) than the WKY rats.

SHR Presented the Same Recognition in Novel Object and Novel Arm Maze Test When Compared to the Age-Matched WKY Rats

In novel object recognition test (Fig. 3a and b), SHR exhibited higher total travel distance (cm) compared to the WKY rats (SHR vs WKY, 2503 vs 1449 cm, p < 0.0001, Fig. 3c). Their ability to recognize novel and familiar objects is as well as that of WKY rats; this ability was quantified through a significance value in the percentage (%) of travel distance in zone (Fig. 3d) and percentage (%) of travel time in zone (Fig. 3e).

In Y maze test (Fig. 4a and b), SHR also exhibited a higher total travel distance (cm) compared to the WKY rats (SHR vs WKY, 3146 vs 2004 cm, p < 0.05, Fig. 4c). Their ability to differentiate between novel arm C and familiar arm B was as well as that of WKY rats; this ability was measured by a significant value in the percentage (%) of travel distance in arms (Fig. 4d) and the percentage (%) of travel time in arms (Fig. 4e).

Fig. 4.

Fig. 4

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The Y maze recognition test data in 58-week-old spontaneously hypertensive rats (SHR, n = 8) and Wistar-Kyoto (WKY, n = 11) rats (a). The moving track of WKY control rat and SHR (b) at T2 is shown. The total travel distance [cm] (c) is shown for the indicated group. Data are presented as the mean ± SEM. *p < 0.05 SHR vs WKY (t-test). The percentage [%] of travel distance in familiar arm B and novel arm C (d) and % of travel time in in arm B and C (e) are shown for the indicated group. *p < 0.05, **p < 0.01 represents the significant difference in arm B and C within same group (t-test)

An Increase of Glutamatergic Receptors was Found in the Hippocampus of Aged Hypertensive rats

In SHR, a significant increase of GluA1 and GluN2B (Fig. 5a, b) in hippocampus was found when compared to the WKY rats. While there were no significant difference in the expression of GluN2A, PSD-95, and GluA3 (Fig. 5a, b) in SHR’s hippocampus. In addition, the expression of glutamatergic receptors and PSD-95 in mPFC (Fig. 6) were not different between SHR and WKY rats.

Fig. 5.

Fig. 5

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The Western blot analysis in hippocampus of 58-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Data show the (a) hippocampus immunoblotting signals of WKY rat and SHR. (b) the statistics results of GluN2A, GluA1, PSD-95, GluN2B, GluA3, and astrocyte (GFAP) in hippocampus (n ≥ 4). Data are presented as the mean ± SEM. **p < 0.01 vs WKY control rat (t-test)

Fig. 6.

Fig. 6

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The Western blot analysis in medial prefrontal cortex (mPFC) of 58-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Data show the (a) mPFC immunoblotting signals of WKY rat and SHR. b the statistics results of GluN2A, GluA1, PSD-95, GluN2B, GluA3, and astrocyte (GFAP) (n ≥ 4). Data are presented as the mean ± SEM. *p < 0.05 vs WKY control rat (t-test)

The immunofluorescence staining (Fig. 7a) showed that the expression of GluN2B in the hippocampus CA3 was significantly higher in SHR than in WKY rat (Fig. 7b). The white arrows indicate neuronal cells with higher levels of GluN2B expression in CA3.

Fig. 7.

Fig. 7

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The expression of neuronal cells (NeuN, red) and GluN2B (green) in hippocampus CA3 in 58-week-old spontaneously hypertensive rats (SHR, n = 4) and Wistar-Kyoto (WKY, n = 4) control rats. Figures show the a NeuN and NR2B immunofluorescence staining. Magnification = 20X; scale bar = 50 μm. The white arrows indicate the neuronal cells with higher GluN2B expression. b NeuN and GluN2B immunofluorescence statistics of WKY rat and SHR in hippocampus CA3. Data are presented as the mean ± SEM (t-test)

A Decrease of Astrocyte was Found in Synaptic Membrane in the Brain of Aged Hypertensive Rats

We evaluated the expression of astrocyte in the brain by using Western blot (Fig. 5) and immunofluorescence staining (Fig. 8), and calculated the immunofluorescence intensity of GFAP and GFAP-positive cells in unit area of brain slice (Fig. 8). Although, no significant changes of astrocyte (GFAP) expression were noted in SHR's hippocampus CA1, CA3, and dentate gyrus (DG) and mPFC by the immunofluorescence staining of brain slices (Fig. 8), the analysis of synaptic membrane of brain in Western blot showed significant decrease of astrocyte in hippocampus (Fig. 5a, b) and mPFC (Fig. 6a, b) in SHR compared to WKY. Thus, we further analyzed the glutamate reuptake transporter, EAAT2, which is expressed on astrocyte. The results of Western blot showed that there was no statistical difference in the expression of EAAT2 in the synaptic membrane between SHR and WKY (Fig. 9). In addition, because EAAT2 was abundantly expressed in tissue fluorescent staining, we directly displayed the image of EAAT2/GFAP in the result (Fig. 10).

Fig. 8.

Fig. 8

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The expression of astrocyte (GFAP) in hippocampus (CA1, CA3 & DG) and medial prefrontal cortex (mPFC) in 58-week-old spontaneously hypertensive rats (SHR, n = 4) and Wistar-Kyoto (WKY, n = 4) control rats. Figures show the (a) GFAP immunofluorescence staining. Magnification = 20X; scale bar = 50 μm. b GFAP immunofluorescence statistics of WKY rat and SHR in CA1, CA3, DG, and mPFC. Data are presented as the mean ± SEM (t-test)

Fig. 9.

Fig. 9

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The Western blot analysis of EAAT2 in medial prefrontal cortex (mPFC) and hippocampus of 58-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Data show the a mPFC and hippocampus immunoblotting signals of WKY rat and SHR. b the statistics results of EAAT2 (n = 4). Data are presented as the mean ± SEM (t-test)

Fig. 10.

Fig. 10

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The expression of excitatory amino acid transporter 2 (EAAT2) and astrocyte (GFAP) in hippocampus and medial prefrontal cortex (mPFC) in 58-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) control rats. a, b show the EAAT2 immunostaining in hippocampus. c show the EAAT2 immunostaining in mPFC. Magnification = 4X & 20X; scale bar = 200 & 50 μm

A Decrease of Microglia Cells was Found in the Brain of Aged Hypertensive Rats

The expression of microglia cells in mPFC and hippocampus CA1, CA3, and DG were evaluated by Iba1 staining. The immuno-intensity of Iba1 and Iba1-positive cells in unit area of brain slice were calculated. We found that the expression of microglia cells in hippocampus CA3 (Fig. 11) in SHR were significantly downregulated compared to the WKY rats. However, there were no significant difference of microglia cells expression in hippocampus CA1 and DG and mPFC between SHR and WKY rats.

Fig. 11.

Fig. 11

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The expression of microglia (IbA1) in hippocampus (CA1, CA3, & DG) and medial prefrontal cortex (mPFC) in 58-week-old spontaneously hypertensive rats (SHR, n = 4) and Wistar-Kyoto (WKY, n = 4) control rats. a IbA1 immunostaining. Magnification = 20X; scale bar = 50 μm. b IbA1 immunostaining statistics of WKY rat and SHR in CA1, CA3, DG, and mPFC. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 vs WKY control rats (t-test)

Discussion

Our data showed that there was no significant difference in novel object and space recognition between aged SHR and aged WKY rats. However, significant hyperactivity behavior was found in aged SHR in open field, novel object recognition, and Y maze test. In addition, significant increasing of glutamatergic receptors GluA1 and GluN2B of hippocampus was found. These findings indicate a possibility that the mechanism of hypertension induces irritability, hyperactivity, and an increase in the expression of excitatory amino acid receptors in hippocampus.

Studies have shown that emotional irritability was associated with increased blood pressure in human subjects (Deter et al. 2006; Benjamin et al. 2012). In rodents, both experimentally and spontaneously hypertensive rats showed a significant higher locomotor activity (Tsai and Lin 1988; Fasmer and Johansen 2016). Thus, the increase of emotional irritability and hyperactivity in chronic hypertension should be closely noticed. The study of the mechanism of hyperactivity caused by chronic hypertension can provide references for pharmacological treatment. Although, studies have shown that an increase of glutamatergic excitatory input to the pre-sympathetic neurons in the PVN of the hypothalamus leads to the increase of sympathetic outflow in various animal models, (Tsuda 2013; Li and Pan 2017) the exact roles of excitatory amino acid system in hypertension induces locomotor hyperactivity is currently not well understood. In this study, we first present the evidences of hippocampus GluA1 and GluN2B elevation and locomotor hyperactivity in 58-week-aged SHR. SHR is a commonly used model of hypertension as these animals develop increase in blood pressure beginning at 6–7 weeks of age and reache a stable level of hypertension by 17–19 weeks of age (Reckelhoff et al. 2018). Previous study had showed that in 12-week-young adult SHR, there were no significant changes of GluN2A and GluN2B in hippocampus (Jensen et al. 2009). However, our data reveal a correlation among chronic high blood pressure, locomotor hyperactivity, and increase of hippocampus glutamatergic receptors in 58-week-aged SHR. Thus, we assumed that the changing of glutamatergic receptors in hippocampus in the older rats was the aftermath of chronic exposure to high blood pressure.

Furthermore, a decrease of astrocyte was found in synaptic membrane of mPFC and hippocampus. As synaptic membrane of neurons may also include neuroglial units, the interrelation between astrocytes with the nerve terminal may explain the possible impact of astrocyte in synapse. As we know, astrocytes reuptake and regulate the amounts of glutamate in brain (Anderson and Swanson 2000); a decrease of astrocyte induces over-excitation of neuronal cells (Hacimuftuoglu et al. 2016). Although we did not find significant changes in the expression of astrocytes and EAAT2 in the immunostaining of brain tissue in SHR, the down-regulation of astrocytes in the synaptic membrane fraction indicated that the reuptake of glutamate in the SHR brain was poor. In addition, due to experimental differences and limitations, it is necessary to further clarify the expression and role of astrocytes in SHR’s brain.

In human subjects, it was found that the hippocampus of adolescents with ADHD was larger than that of the control group (Plessen et al. 2006). Previous studies have shown that hyperactivity of hippocampus may regard as an early sign of psychosis in human (Maureen McHugo et al. 2019; Bakker et al. 2012). Thus, the abnormal activation of hippocampus may be highly correlated to the progression of psychiatric and neurologic disorder. We hypothesize that the increase of glutamatergic activity in hippocampus may over-activate the hippocampus and be involved in causing the subsequent abnormal behaviors, such as emotional irritability and hyperactivity. These findings remind us that understanding the mechanism of synaptic changes of chronic hypertension is an important issue in hypertension-induced neuronal dysfunction.

Interestingly, in our elderly SHR, long-term high blood pressure lead to a significant reduction of microglia in mPFC and hippocampus. This is the first report that represented the expression of microglia in aged SHR’s mPFC and hippocampus. In previous studies, the activated microglia cells were found in rodents’ PVN with hypertensive situation (Shi et al. 2010; Winklewski et al. 2015). In young adult rat with chronic angiotensin II infusion model, the activation of microglial cells and the generation of proinflammatory cytokines in the PVN of hypothalamus were found (Shi et al. 2010). The activation of microglia cells in PVN was considered contributing to the formation of neurogenic hypertension (Shi et al. 2010; Winklewski et al. 2015). In addition, a controversial finding has been proposed that PVN microglia activation is not related to the maintenance of hypertension in 15-week-old SHR (Takesue et al. 2017). Our data resembled that of another study which states that in the rostral of ventrolateral medulla of SHR, the expression of microglia cells was downregulated (Cohen et al. 2019). Thus, we suggest that no significant neuronal inflammation were found in mPFC and hippocampus in aged and hyperactivity SHR, and that down-regulation of brain immune system in chronic hypertension should be noticed. The different effects of short-term and long-term exposure of hypertension on the expression of microglia cells in brain are interesting and needs to be further studied.

In this study, the 58-week-old SHR showed the same recognition and explorative ability in novel objects and novel arm maze test as the age-matched WKY rat. And the expression of NeuN in hippocampus CA3 were not different between SHR and WKY rat. Motivational tests such as the alternative maze test and novel object recognition test were used to measure cognition due to the limitation of aged rats. Although such tests cannot totally represent the cognitive function of rat and their function of hippocampus, our data also show the same CA3 neuronal cell counts and novelty recognition between SHR and WKY rat thereby further concurring that there is no change in cognition between the groups. In human subjects, studies had showed that hypertension is correlated to the cognitive dysfunction (Brady et al. 2005; Nishtala et al. 2015; Walker et al. 2017; Moraes et al. 2020) later on in life. The uncontrolled hypertension produces specific cognitive deficits in aged people (Brady et al. 2005). Patients with more severe hypertension presented worse performance in executive functioning (Moraes et al. 2020). Therefore, it is hypothesized that neuronal dysfunction may develop when subject is under chronic exposure to higher blood pressure. However, the other study showed that midlife hypertension was not associated with late-life cognitive impairment (Nishtala et al. 2015). Midlife SBP related to the late-life attention and verbal fluency impairments only in subjects with hypertension and ApoE4 genotype (Nishtala et al. 2015). Thus, we suggest that the impacts of higher blood pressure in cognitive decline may need more extensive study.

Taken together, these results indicate the mechanisms underlying the pathogenesis of hypertension and the development of locomotor hyperactivity in aged SHR. The changes of excitatory amino acid receptors may explain the mechanism of irritability or hyper-locomotor activity of chronic hypertension.

Conclusion

Long-term exposure to high blood pressure induces upregulation of glutamatergic receptors in hippocampus. An increase of GluA1 and GluN2B in hippocampus may over-activate the hippocampus and contribute to the hyper-locomotor activity or subsequent neuronal dysfunction of SHR. The changing of glutamatergic system may act as therapeutic targets for hypertension-induced irritability and hyperactivity.

Author Contributions

PS-YY, Y-CL, and S-L Chen performed most of the experiments; C-HC helped with parts of the experiments and revised the manuscript; S-LC and Y-CL designed the study, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by grant M109108 (to SLC) from Kaohsiung Medical University and the MOST Grants #105–2628-B-037–003-MY3 (to SLC) and (to) from the Taiwan Ministry of Science and Technology.

Data Availability

All authors have ensured that all data and materials support the published statement and comply with field standards. The datasets generated during or analyzed during the current study are available from the corresponding author on reasonable request.

Code Availability

The softwares (OLYMPUS cellSens Dimension 1.13., Prism 5, and SPSS 22) that were used in this study have been authorized by the original company.

Declarations

Conflict of interest

None of the authors have any conflict of interest to declare.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

PatrickSzu-Ying Yen, Yen-Chin Liu have contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All authors have ensured that all data and materials support the published statement and comply with field standards. The datasets generated during or analyzed during the current study are available from the corresponding author on reasonable request.

The softwares (OLYMPUS cellSens Dimension 1.13., Prism 5, and SPSS 22) that were used in this study have been authorized by the original company.

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