BDNF shifts excitatory-inhibitory balance in the paraventricular nucleus of the hypothalamus to elevate blood pressure

Abstract

Presympathetic neurons in the paraventricular nucleus of the hypothalamus (PVN) play a key role in cardiovascular regulation. We have previously shown that brain-derived neurotrophic factor (BDNF), acting in the PVN, increases sympathetic activity and blood pressure and serves as a key regulator of stress-induced hypertensive responses. BDNF is known to alter glutamatergic and GABA-ergic signaling broadly in the central nervous system, but whether BDNF has similar actions in the PVN remains to be investigated. Here, we tested the hypothesis that increased BDNF expression in the PVN elevates blood pressure by enhancing N-methyl-d-aspartate (NMDA) receptor (NMDAR)- and inhibiting GABA_A receptor (GABA_AR)-mediated signaling. Sprague-Dawley rats received bilateral PVN injections of AAV2 viral vectors expressing green fluorescent protein (GFP) or BDNF. Three weeks later, cardiovascular responses to PVN injections of NMDAR and GABA_AR agonists and antagonists were recorded under α-chloralose-urethane anesthesia. In addition, expressions of excitatory and inhibitory signaling components in the PVN were assessed using immunofluorescence. Our results showed that NMDAR inhibition led to a greater decrease in blood pressure in the BDNF vs. GFP group, while GABA_AR inhibition led to greater increases in blood pressure in the GFP group compared to BDNF. Conversely, GABA_AR activation decreased blood pressure significantly more in GFP vs. BDNF rats. In addition, immunoreactivity of NMDAR1 was upregulated, while GABA_AR-α1 and K⁺/Cl⁻ cotransporter 2 were downregulated by BDNF overexpression in the PVN. In summary, our findings indicate that hypertensive actions of BDNF within the PVN are mediated, at least in part, by augmented NMDAR and reduced GABA_AR signaling.

NEW & NOTEWORTHY We have shown that BDNF, acting in the PVN, elevates blood pressure in part by augmenting NMDA receptor-mediated excitatory input and by diminishing GABA_A receptor-mediated inhibitory input to PVN neurons. In addition, we demonstrate that elevated BDNF expression in the PVN upregulates NMDA receptor immunoreactivity and downregulates GABA_A receptor as well as KCC2 transporter immunoreactivity.

INTRODUCTION

The paraventricular nucleus (PVN) of the hypothalamus is an important brain region involved in the regulation of autonomic, endocrine, and cardiovascular responses (1, 2). The PVN plays a specific role in cardiovascular regulation via presympathetic neurons projecting to the rostral ventrolateral medulla (RVLM) or directly to spinal sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord (3–6). Increased activity of PVN presympathetic neurons contributes to elevated sympathetic activity and blood pressure in hypertension (7–9), which can increase the risk of other cardiovascular, renal, and cerebrovascular disorders (10, 11). Glutamate and γ-aminobutyric acid (GABA) are the main excitatory and inhibitory neurotransmitters within the PVN and have been shown to alter the firing activity of PVN neurons (12, 13). In addition, increases in excitatory and decreases in inhibitory mechanisms within the PVN are thought to contribute to enhanced sympathetic output in hypertension (14–16). For example, PVN neurons are under significant tonic GABA_A receptor (GABA_AR)-mediated inhibition under normotensive conditions (17, 18), which is reduced leading to elevated sympathetic activity in hypertension and heart failure (19–21). Conversely, NMDA receptor (NMDAR)-mediated excitatory neurotransmission is augmented in several hypertensive animal models including spontaneously hypertensive rats (SHRs) (22), Dahl salt-sensitive rats on high salt diet (23), water-deprived rats (24), or rats subject to heart failure (25).

Brain-derived neurotrophic factor (BDNF) has been shown to be upregulated in the PVN in response to various hypertensive stimuli, such as chronic and acute stress, hyperosmolality, and repeated amphetamine administration (26–30), and our recent studies revealed that BDNF, acting in the PVN, is a key regulator of blood pressure. We have shown that both long-term overexpression and acute microinjection of BDNF in the PVN led to significant increases in blood pressure and heart rate by modulating angiotensin II and catecholaminergic signaling (31–33). On the other hand, inhibition of BDNF-TrκB receptor signaling in the PVN diminished stress-induced hypertensive responses (34).

BDNF is known to modulate glutamatergic and GABA-ergic signaling mechanisms in the central nervous system (CNS) via multiple mechanisms. For example, BDNF increases presynaptic glutamate release by activating synapsins (35) and enhances NMDAR expression and membrane trafficking (36–38), while it reduces the expression and membrane density of GABA_AR (39–41). BDNF has also been shown to downregulate the expression of K⁺/Cl⁻ cotransporter 2 (KCC2) (42–48), which maintains low intracellular chloride concentration and serves to establish the chloride ion gradient in neurons necessary for hyperpolarizing GABA_AR currents (49). However, despite its significant role in regulating excitatory/inhibitory synaptic mechanisms elsewhere in the CNS, actions of BDNF on NMDAR and GABA_AR signaling in the PVN remains to be investigated.

Here, we set out to test the hypothesis that upregulation of BDNF in the PVN elevates blood pressure by altering the excitatory/inhibitory balance in the PVN via diminishing GABA_AR-mediated inhibitory mechanisms and augmenting NMDAR-mediated excitatory mechanisms. We used our previously published model of vector-mediated overexpression of BDNF in the PVN to induce hypertension (31, 33) and investigated the interaction of BDNF with glutamatergic and GABA-ergic signaling mechanisms. Better understanding of how BDNF affects the PVN neurocircuitry, sympathetic activity and ultimately cardiovascular function could aid in the development of novel therapies for the treatment of hypertension.

METHODS

All animal housing, handling, and surgical and experimental procedures were conducted within an Association for the Assessment and Accreditation of Laboratory Care International-accredited animal care facility at the University of Vermont, in accordance with the National Institutes of Health (NIH) Policy on Humane Care and Use of Laboratory Animals and the NIH Guide for the Care and Use of Laboratory Animals. Experiments were performed in male Sprague-Dawley (SD) rats obtained from Charles River (Saint-Constant, QC, Canada). Rats were housed individually with a 12:12-h light-dark cycle (lights on at 6:00 AM), with free access to food (standard chow) and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Vermont.

Experimental Design

Experiment 1.

The aim of this experiment was to determine the effect of BDNF on NMDAR- and GABA_AR-mediated signaling in the PVN. Seven-week-old male SD rats received bilateral PVN injections of adeno-associated viral vectors (AAV2, 10¹² vps/mL, 200 nL/side) expressing GFP or myc epitope-tagged BDNF fusion protein (BDNFmyc). Three weeks later, blood pressure and heart rate responses were recorded following PVN injections of agonists and antagonists of NMDAR and GABA_AR under α-chloralose-urethane anesthesia. At the end of the experiment, animals were deeply anesthetized and perfused transcardially with phosphate buffered saline (PBS) and 4% paraformaldehyde. PVN expressions of GFP, BDNFmyc and drug injection sites (indicated by a red fluorescent microbead solution) were verified with fluorescent microscopy in coronal sections of the PVN (Fig. 1).

Figure 1.

Representative fluorescent images of a coronal brain sections ∼1.8 mm posterior to bregma showing PVN expression of GFP (A), BDNFmyc (B), and the red fluorescent microbead solution that was mixed with injected drug solution to verify injection sites (C). D: diagrams of the PVN ∼1.8 mm and ∼1.9 mm posterior to bregma showing locations of drug injections in rats previously injected with AAV2-GFP (indicated by circles on the left side of the diagram) or AAV2-BDNFmyc (indicated by squares on the right side of the diagram). E: average number of GFP- and myc-positive cells in subnuclei of the PVN; dorsal parvocellular nuclei (DP), medial parvocellular nuclei (MP), ventrolateral parvocellular nuclei (VLP), and posterior magnocellular nuclei (PM). F: changes in mean arterial pressure (MAP; top) and heart rate (HR; bottom) in response to PVN injections of artificial cerebrospinal fluid (aCSF), the vehicle for all drug injections. G: verification of the specificity of secondary antibodies and the lack of bleed-through from green to red channel. Scale bars: 250 µm. 3 V, third ventricle; AAV2, adeno-associated viral vectors; BDNF, brain-derived neurotrophic factor; BDNFmyc, myc epitope-tagged BDNF; GFP, green fluorescent protein; PVN, paraventricular nucleus.

Experiment 2.

The goal of this experiment was to determine the effect of BDNF on protein expressions of various components of inhibitory and excitatory signaling mechanisms in the PVN. Seven-week-old male SD rats received bilateral PVN injections of AAV2 viral vectors (10¹² vps/mL, 200 nL/side) expressing GFP or BDNFmyc. Three weeks later, animals were deeply anesthetized and perfused transcardially with phosphate buffered saline (PBS) and 4% paraformaldehyde. PVN expressions of NMDAR1, GABA_AR-α1 subunit, and KCC2 were assessed using immunofluorescence and confocal microscopy.

Surgical Procedures

Surgeries were performed using aseptic techniques under continuous isoflurane anesthesia (5% induction, 2%–3% maintenance) delivered in oxygen. Depth of anesthesia was ensured by lack of a reflex response to pinch of the hindpaw. Carprofen (5 mg·kg^–1·day^–1 sc) was used for postsurgical analgesia administered at the beginning of surgery and for 2 days after surgery. Rats were put under isoflurane anesthesia and placed in a stereotaxic frame, AAV2 viral vectors (10¹² viral particles/mL, 200 nL/side) were injected bilaterally into the PVN using pipettes pulled from thin walled borosilicate glass capillary tubes (OD, 1 mm; ID, 0.58 mm; tip diameter: ∼25 µm; World Precision Instruments Inc., Sarasota, FL) at the following stereotactic coordinates: 1.80 mm posterior to bregma, 1.70 mm lateral to the midline, and 7.65 mm ventral from the dorsal surface of the brain, with the micropipette tilted 10° laterally toward the midline. Virus stocks were injected over 5 min using a pneumatic pico pump (World Precision Instruments Inc., Sarasota, FL). The pipette was left in place for an additional 3 min before being withdrawn.

Assessment of Cardiovascular Responses to NMDAR and GABA_AR Agonists and Antagonists

The left femoral artery and vein were catheterized under isoflurane anesthesia 3 weeks after bilateral PVN injections of AAV2-GFP or AAV2-BDNFmyc. Rats were then placed in a stereotaxic frame, and isoflurane anesthesia was gradually switched to intravenous α-chloralose (60 mg × kg⁻¹ × h⁻¹) and urethane (800 mg × kg⁻¹ × h⁻¹) anesthesia administered through the femoral vein catheter over a 30-min period, during which, isoflurane was gradually reduced from 2.5% to 0%. Blood pressure, heart rate, toe-pinch, and eye blink reflexes were monitored closely to ensure the animal remained anesthetized. After complete withdrawal of isoflurane, anesthesia was maintained by intravenous α-chloralose (15 mg × kg⁻¹ × h⁻¹) and urethane (200 mg × kg⁻¹ × h⁻¹) infusion for the remainder of the experiment. After establishment of steady baseline blood pressure and heart rate for a minimum of 30 min, rats received bilateral PVN injections of one of the following drugs: NMDA (100 µM, 200 nL/side, Sigma-Aldrich, St. Louis, MO; M3262) (50), AP5 (10 mM, 200 nL/side; Tocris, Minneapolis, MN; #0106) (15, 22), muscimol (10 mM, 200 nL/side, Sigma-Aldrich, St. Louis, MO; G019) (51), or gabazine (2 mM, 200 nL/side, Tocris, Minneapolis, MN; #1262) (22). Blood pressure was monitored via the catheter placed in the left femoral artery, and heart rate was extracted from the pulsatile pressure wave by using Laboratory Chart Pro 8 (ADInstruments, Colorado Springs, CO). All drugs were diluted in artificial cerebrospinal fluid (aCSF), which had no effect on either arterial blood pressure or heart rate when injected into the PVN (Fig. 1F). To verify locations of drug injections in the PVN with fluorescent microscopy, 10% rhodamine-labeled fluorescent microspheres (0.04 µm; Molecular Probes, Eugene, OR) were mixed into the injection solution.

Viral Vector-Mediated Gene Transfer into the PVN

AAV2 viral vectors were used to elicit the expression of enhanced GFP and BDNFmyc, derived from rat bdnf, constructed, and packaged by Vector Biolabs (Philadelphia, PA). The expression of GFP and BDNFmyc was driven by a chicken beta-actin promoter with human cytomegalovirus enhancer and a woodchuck posttranscriptional regulatory element, which enhanced the expression of transgenes present downstream of GFP and BDNFmyc. The BDNFmyc plasmid was a generous gift from Dr. Ronald Klein (LSU Health Sciences Center, Shreveport, LA) and was used previously to protect retinal ganglion cells in a rat glaucoma model (52), and to study cardiovascular effects of BDNF in the PVN (31, 33). In addition, full efficacy of BDNFmyc expression driven by the rat neuron-specific enolase promoter was confirmed previously both in vitro and in vivo (53).

Analysis of Cardiovascular Data from Anesthetized Animals

Blood pressure and heart rate were recorded using LabChart software version 8.0.7 (ADInstruments, Colorado Springs, CO) at a 1,000-Hz sampling rate and condensed to 15- or 30-s moving averages for data analysis and presentation. Maximum and average changes in mean arterial pressure (MAP) and heart rate following drug microinjections were evaluated.

Immunofluorescence

After experiments 1 and 2, animals were perfused with 400 mL of ice-cold PBS followed by 400 mL of ice-cold 4% paraformaldehyde in PBS. Brains were removed and post-fixed for 2 h in 4% paraformaldehyde and then equilibrated in 30% sucrose solution at 4°C. Coronal sections (40 µm) were cut on a freezing microtome (Leica SM2000R) and mounted on Fisher Superfrost Plus slides. BDNFmyc, NMDAR1, GABA_AR-α1 and KCC2 were detected using the following primary antibodies (1:200, overnight incubation at 4°C): anti-c-Myc (Santa Cruz Biotechnology, Dallas, TX; 9E10), anti-c-Myc (Abcam, Cambridge, MA; ab9106) anti-NMDAR1 (Novus Biologicals, R1JHL), anti-GABA_AR-α1 (Abcam, Cambridge, MA; ab33299), and anti-KCC2 (Sigma-Aldrich, St. Louis, MO; MABN88). Secondary antibodies (1:200, 2-h incubation at room temperature) were: donkey anti-mouse A546 and donkey anti-rabbit A555 (Invitrogen, Waltham, MA). GFP, BDNFmyc and fluorescent tracer were detected with a fluorescent microscope (Nikon Eclipse 50i); NMDAR, GABA_AR-α1 and KCC2 immunofluorescences were assessed with a scanning confocal microscope (Nikon A1R).

The NMDAR1, GABA_AR-α1, and KCC2 primary antibodies have been verified in previous publications. The NMDAR1 primary antibody has been tested using immunofluorescence (54) and immunocytochemistry (55) in rat spinal cord sections and neurons, immunohistochemistry in mouse hippocampus sections (56), and using immunocytochemistry in human T-lymphocytes (55). The GABA_AR-α1 primary antibody has been previously tested using immunofluorescence in the somatosensory cortex of mice (57), using immunostaining in projection neurons in the telencephalon of chickens (58), and immunohistochemistry (59), and immunocytochemistry (60) in mouse hippocampal sections and neurons. The KCC2 primary antibody has been tested using immunofluorescence in mouse PVN sections (61), and by Western blot analysis in the cortex, hippocampus, and sub-cortex of mice (62). We tested the secondary antibodies for non-specific binding and found there was no staining from the secondary antibodies without application of the primary antibody, and that there was no bleed-through from green to red channel (Fig. 1G).

Image Analysis

Z-stack images with 16-bit depth were obtained using a Nikon A1R confocal laser-scanning system from PVN brain slices labeled with immunofluorescence as described above. The z-stacks were then deconvoluted using the deconvolution software AutoQuant X3 (Media Cybernetics, Inc., Rockville, MA). Following this, the green channel of the z-stack representing GFP or BDNFmyc expression, was opened in ImageJ and thresholded using the Phansalkar Auto Local Threshold Algorithm (63). This procedure uses a combination of local mean, local standard deviation and normalization to set a threshold below the local mean to select the foreground, reject the background and boost the threshold at lower intensities (64). In addition, the “Remove Outliers” function in ImageJ was used to further improve image masking based on GFP and BDNFmyc expression. Finally, the “Analyze Particles” function was used to generate regions of interest (ROI) representing neurons expressing BDNFmyc or GFP. Using these ROIs generated in the green channel, mean fluorescence intensity (ROI mean) and maximum fluorescence intensity (ROI max) were measured in the red channel representing expressions of one of the target proteins (NMDAR1, GABA_AR-α1 or KCC2). In addition to ROI-based analysis, mean fluorescence intensity within the whole PVN was also calculated. Analysis was conducted separately for each side of the PVN, and the mean calculated within each experimental group. Samples from control and treatment groups were processed, imaged and analyzed under the same conditions.

Statistics

Statistical tests were performed using Prism 8.2 software (GraphPad, San Diego, CA). Maximum decreases or increases in MAP and heart rate during microinjection experiments and fluorescent intensities in the immunofluorescent experiments were compared between GFP and BDNF groups using unpaired t test. Results are expressed as means ± SE, and the criterion for statistical significance was P < 0.05.

RESULTS

Effect of BDNF on NMDAR- and GABA_AR-Mediated Signaling

Analysis of NMDAR-mediated cardiovascular responses showed that inhibition of NMDAR with AP5 significantly lowered MAP and heart rate in the BDNF group, while the same treatment had no effect in the GFP group indicating an elevated glutamatergic tone in BDNF rats. Maximum decreases in blood pressure and heart rate in response to AP5 were 16.5 ± 5.0 mmHg and 37 ± 11 beats/min in the BDNF group, compared with 2.6 ± 1.1 mmHg (P < 0.05) and 6 ± 3 beats/min (P < 0.05) in the GFP group (Fig. 2, A and B). Activation of NMDAR with NMDA injections induced a biphasic MAP response resulting in a delayed increase in MAP in the GFP group and a significant initial decline followed by a return back to baseline MAP in the BDNF group (Fig. 2C). In contrast, heart rate fell in both GFP and BDNF rats in response to NMDA injections. Maximum initial decreases in MAP were 3.6 ± 2.1 mmHg in the GFP and 12.6 ± 3.1 mmHg in the BDNF group (P < 0.05). Maximum increases in MAP were 7.4 ± 1.2 mmHg in the GFP and 1.2 ± 0.9 mmHg in the BDNF group (P < 0.05). Maximum heart rate decreases were 20 ± 5 beats/min in the GFP and 23 ± 4 beats/min in the BDNF group (Fig. 2D).

Figure 2.

Changes in mean arterial pressure (MAP; top) and heart rate (HR; bottom) in response to PVN injections of the NMDAR antagonist AP5 (A and B) and NMDAR agonist NMDA (C and D) in rats previously treated with AAV2-GFP or AAV2-BDNFmyc. A: MAP and HR obtained with 15-s moving average over 5 min following AP5 injection in GFP (n = 5) and BDNFmyc (n = 7) treated rats. B: maximum decreases in MAP and heart rate to AP5 injections. C: MAP and HR obtained with 30-s moving average over 10 min following NMDA injection in GFP (n = 5) and BDNFmyc (n = 7) treated rats. D: maximum decreases and increases in MAP and maximum decreases in heart rate in response to NMDA injections. Results represent means ± SE, statistical analysis was done on maximum changes in MAP and heart rate. *P < 0.05 for GFP vs. BDNF. AAV2, adeno-associated viral vectors; BDNF, brain-derived neurotrophic factor; BDNFmyc, myc epitope-tagged BDNF; GFP, green fluorescent protein; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; PVN, paraventricular nucleus.

Analysis of GABA_AR-mediated cardiovascular responses indicated that MAP elevation following GABA_AR inhibition with gabazine was significantly reduced in the BDNF group compared with GFP indicating a diminished GABAergic tone in BDNF rats. In response to gabazine, maximum average increases in MAP and heart rate were 32.7 ± 6.9 mmHg and 70 ± 19 beats/min in the BDNF group, compared with 70 ± 12 mmHg (P < 0.05) and 132 ± 19 beats/min (P < 0.05) in the GFP group (Fig. 3, A and B). In contrast, activation of GABA_AR with muscimol decreased MAP significantly more in the GFP group compared to the BDNF group indicating a reduced sensitivity to GABA_AR activation in BDNF rats. Muscimol-induced maximum decreases in MAP and HR were 14.6 ± 1.6 mmHg and 19 ± 4 beats/min in the BDNF group, compared with 25.4 ± 4.8 mmHg (P < 0.05) and 42 ± 6 beats/min (P < 0.01) in the GFP group (Fig. 3, C and D).

Figure 3.

Changes in mean arterial pressure (MAP; top) and heart rate (HR; bottom) in response to PVN injections of the GABA_AR antagonist gabazine (A and B) and GABA_AR agonist muscimol (C and D) in rats previously treated with AAV2-GFP or AAV2-BDNFmyc. A: MAP and HR obtained with 30-s moving average over 10 min following gabazine injection in GFP (n = 6) and BDNFmyc (n = 6) treated rats. B: maximum increases in MAP and heart rate to gabazine injections. C: MAP and HR obtained with 30-s moving average over 15 min following muscimol injection in GFP (n = 5) and BDNFmyc (n = 7) treated rats. D: maximum decreases in MAP and heart rate in response to muscimol injections. Results represent means ± SE, statistical analysis was done on maximum changes in MAP and heart rate. *P < 0.05, **P < 0.01 for GFP vs. BDNF. AAV2, adeno-associated viral vectors; BDNF, brain-derived neurotrophic factor; BDNFmyc, myc epitope-tagged BDNF; GFP, green fluorescent protein.

BDNF-Induced Changes in the Expression of Excitatory and Inhibitory Signaling Components

NMDAR1 expression was first quantified in GFP- and BDNFmyc-expressing PVN neurons by analyzing mean and maximum fluorescence intensity representing anti-NMDAR1 antibody binding in ROIs generated using GFP and BDNFmyc fluorescence. Mean and maximum NMDAR1 fluorescence were both found to be significantly elevated in BDNFmyc-expressing neurons compared to GFP-expressing neurons, and mean NMDAR1 fluorescence intensity within the whole PVN was also found to be significantly elevated in the BDNF group compared to the GFP group (P < 0.05, Fig. 4I). High-magnification images also revealed increases in both bright, punctate NMDAR1 fluorescence around BDNFmyc-expressing neurons and in diffuse intracellular NMDAR1 immunoreactivity (Fig. 4, G and H).

Figure 4.

Representative fluorescent images indicating immunoreactivity of NMDAR1 (red channel) and its overlap with GFP (B and C, green channel) and BDNFmyc (F and G, green channel) in the PVN of rats previously injected with AAV2-GFP (n = 8) and AAV2-BDNF (n = 6). Images were taken at ×20 magnification (A, B, E, and F, scale bar: 200 µm) and at ×40 magnification with ×2 zoom (C, D, G, and H, scale bar: 25 µm). Outline of a few representative in-focus GFP- and BDNFmyc-positive cells are indicated with dashed lines for reference (D and H). Mean NMDAR1 fluorescent intensity within cell ROIs, maximum NMDAR1 fluorescent intensity within cell ROIs and mean NMDAR1 fluorescence intensity in the whole PVN were averaged (I). Results represent means ± SE. *P < 0.05, **P < 0.01 for GFP vs. BDNF. AAV2, adeno-associated viral vectors; BDNF, brain-derived neurotrophic factor; BDNFmyc, myc epitope-tagged BDNF; GFP, green fluorescent protein; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; PVN, paraventricular nucleus; ROI, region of interest.

In contrast with NMDAR1, GABA_A-α1 immunoreactivity was found to be inhibited by BDNF in the PVN. While mean GABA_A-α1 immunofluorescence was not significantly different between GFP- and BDNFmyc-expressing neurons (P = 0.40), maximum GABA_A-α1 immunofluorescence was significantly reduced within ROIs of the BDNF group compared to GFP (P < 0.05), and mean GABA_A-α1 immunofluorescence intensity within the whole PVN was also significantly lower in BDNF rats compared to GFP (P < 0.05, Fig. 5I). High-magnification images also indicated a loss of bright fibrous and punctate GABA_A-α1 immunofluorescence around BDNFmyc-positive neurons (Fig. 5, G and H).

Figure 5.

Representative fluorescent images indicating immunoreactivity of GABA_AR-α1 (red channel) and its overlap with GFP (B and C, green channel) and BDNFmyc (F and G, green channel) in the PVN of rats previously injected with AAV2-GFP (n = 7) and AAV2-BDNF (n = 7). Images were taken at ×20 magnification (A, B, E, and F, scale bar: 200 µm) and at ×40 magnification with ×2 zoom (C, D, G, and H, scale bar: 25 µm). Outline of a few representative in-focus GFP- and BDNFmyc-positive cells are indicated with dashed lines for reference (D and H). Mean GABA_AR-α1 fluorescent intensity within cell ROIs, maximum GABA_AR-α1 fluorescent intensity within cell ROIs and mean GABA_AR-α1 fluorescence intensity in the whole PVN were averaged (I). Results represent means ± SE. *P < 0.05 for GFP vs. BDNF. AAV2, adeno-associated viral vectors; BDNF, brain-derived neurotrophic factor; BDNFmyc, myc epitope-tagged BDNF; GFP, green fluorescent protein; PVN, paraventricular nucleus; ROI, region of interest.

As KCC2 plays an important role in neurons establishing chloride ion gradient necessary for GABA-mediated inhibition, we also analyzed the effect of BDNF overexpression on KCC2 immunofluorescence. We found that mean and maximum KCC2 immunofluorescence within BDNFmyc expressing neurons were significantly reduced compared with GFP-expressing neurons (P < 0.01), and that mean fluorescence intensity within the whole PVN mean was also significantly lower in the BDNF group compared with GFP (P < 0.01, Fig. 6I). In addition, high-magnification images indicated a loss of bright fibrous and punctate KCC2 immunofluorescence around BDNFmyc-positive neurons (Fig. 5, G and H).

Figure 6.

Representative fluorescent images indicating immunoreactivity of KCC2 (red channel) and its overlap with GFP (B and C, green channel) and BDNFmyc (F and G, green channel) in the PVN of rats previously injected with AAV2-GFP (n = 8) and AAV2-BDNF (n = 8). Images were taken at ×20 magnification (A, B, E, and F, scale bar: 200 µm) and at ×40 magnification with ×2 zoom (C, D, G, and H, scale bar: 25 µm). Outline of a few representative in-focus GFP- and BDNFmyc-positive cells are indicated with dashed lines for reference (D and H). Mean KCC2 fluorescent intensity within cell ROIs, maximum KCC2 fluorescent intensity within cell ROIs and mean KCC2 fluorescence intensity in the whole PVN were averaged (I). Results represent means ± SE. **P < 0.01 for GFP vs. BDNF. AAV2, adeno-associated viral vectors; BDNF, brain-derived neurotrophic factor; BDNFmyc, myc epitope-tagged BDNF; GFP, green fluorescent protein; PVN, paraventricular nucleus; ROI, region of interest.

DISCUSSION

BDNF is upregulated in the PVN in response to hypertensive stimuli such as chronic and acute stress, hyperosmolality, and repeated amphetamine administration (26–30). In addition, when acutely injected or overexpressed in the PVN, BDNF significantly increases blood pressure and heart rate (31–33). We have previously shown that these hypertensive actions of BDNF are partially mediated by an interaction with angiotensin II (Ang II) signaling and by downregulating beta-adrenergic receptor-mediated hypotensive mechanisms in the PVN (31–33). With the current study, we extend these observations by demonstrating that BDNF also promotes a shift in excitatory/inhibitory balance in the PVN by augmenting NMDAR signaling and inhibiting GABA_AR signaling.

BDNF has been shown to enhance excitatory and diminish inhibitory neurotransmission in multiple regions of the CNS including the hippocampus, where these BDNF-mediated actions are essential for synaptic plasticity, learning and memory (65–70). A similar BDNF-mediated shift in excitatory/inhibitory balance towards excitation in the PVN could contribute to increasing sympathetic activity and may be an important component in elevating blood pressure in various hypertensive models. For example, presympathetic PVN neurons in SHRs receive significantly enhanced glutamatergic tone and demonstrate markedly diminished GABA_AR-mediated inhibition or even a reversal of inhibition to excitation (20, 22). Similarly, elevated blood pressure in Dahl salt-sensitive rats on high salt diet (23), in water-deprived rats (24), or in rats subject to heart failure (25) or chronic intracerebroventricular infusion of Ang II (71) depend on increased glutamatergic tone in the PVN, enhanced NMDAR expression and NMDA-mediated hypertensive responses. In contrast, NMDAR1 deletion in the PVN results in a significant attenuation of Ang II-induced blood pressure increases (72).

In correlation with these studies, we found that BDNF overexpression similarly augments PVN glutamatergic transmission and diminishes GABA_AR-mediated inhibition. For example, NMDAR1 immunoreactivity in PVN neurons was significantly upregulated by BDNF based on multiple image analytic methods, and blockade of NMDA-receptors with AP5 significantly lowered MAP and heart rate in BDNF rats, whereas the same treatment had no effect in the GFP group. This indicates that in contrast with normotensive animals, rats subject to BDNF overexpression in the PVN demonstrate a significant elevation of glutamate–NMDA-mediated excitatory tone. However, further activation of NMDAR with microinjections of NMDA produced somewhat contradictory responses. In GFP rats, after a transient dip, blood pressure increased in response to NMDA, whereas in BDNF rats, the initial decreases in blood pressure were significantly larger compared to those in GFP rats, and while blood pressure increased following this initial response, it failed to significantly rise above baseline. l-glutamate or NMDA injections into the PVN have typically been found to increase blood pressure, and sympathetic nerve activity in both anesthetized and conscious normotensive rats (14, 50, 73–75). However, there is some disagreement in the literature, since PVN glutamate injections in anesthetized SD rats led to no changes in blood pressure and a decrease in heart rate (76), while in anesthetized Wistar rats, glutamate decreased blood pressure with no change in heart rate (77). Taken together, based on our results, we suggest that following BDNF overexpression, PVN presympathetic neurons receive significantly augmented glutamatergic NMDAR-mediated input as indicated by our NMDAR inhibitor experiments. This elevated glutamatergic tone is potentially caused by enhanced presynaptic glutamate release and/or upregulated postsynaptic NMDAR1 expression and signaling. Furthermore, due to elevated baseline NMDA signaling, additional stimulation of NMDAR with NMDA microinjections may have led to synaptic depression (78) in BDNF-treated rats leading to reduced firing of PVN presympathetic neurons and an enhanced initial decrease in blood pressure. Similar actions by BDNF have been previously described in hippocampal neurons. Presynaptically, BDNF stimulates glutamate release leading to an increase in the frequency of miniature excitatory postsynaptic currents (79, 80), whereas postsynaptically, BDNF enhances NMDAR1 expression, membrane density, phosphorylation and opening probability of the NMDAR (36, 81–84). Alternatively, it is also possible that exogenous NMDA activates inhibitory neurons, and BDNF treatment augments this effect leading to enhanced inhibition of PVN presympathetic neurons.

In terms of BDNF actions on inhibitory neurotransmission in the PVN, we found that blood pressure and heart rate elevations following GABA_AR blockade were significantly reduced by BDNF overexpression in the PVN indicating a BDNF-induced decline in GABAergic tone. Conversely, activation of GABA_AR with muscimol resulted in significantly reduced hypotensive and bradycardic responses in the BDNF group compared with GFP indicating that mechanisms downstream of GABA_AR were also blunted by BDNF. Interestingly, these results demonstrating a profound BDNF-mediated effect on GABA_AR-mediated inhibition in the PVN correlate well with those reported in SHRs, where PVN injections of the GABA_AR agonists isoguvacine or muscimol have been found to reduce blood pressure more significantly in normotensive WKY and SD rats compared to SHRs (51, 85). Meanwhile PVN microinjections of the GABA_AR antagonist, gabazine or bicuculline, have been found to increase blood pressure significantly more in normotensive rats compared to SHRs (14, 20, 51, 85). In addition, GABA_AR expression and binding sites are reduced in the PVN of SHRs compared to control rats (86, 87), as is the frequency of GABAergic inhibitory postsynaptic currents of presympathetic neurons (20, 88).

Effects of BDNF on GABAergic signaling throughout the CNS are well-documented. For example, acute BDNF application in the hippocampus has been shown to reduce evoked and spontaneous GABAergic currents (89, 90) and facilitate the early phase of long-term potentiation via TrκB receptor signaling (91–93). In addition, BDNF has been shown to mediate GABA_AR−α1 downregulation associated with epilepsy (41), and control GABA_AR-α4 expression in hippocampal neurons (94). In this study, we quantified GABA_AR-α1subunit immunoreactivity in the PVN and found that maximum GABA_AR-α1 immunofluorescence level within ROIs representing BDNFmyc expressing neurons was significantly reduced compared to GFP-expressing neurons in control rats. However, mean GABA_AR-α1 immunofluorescence level within ROIs was similar in the two groups. The difference between these two parameters may indicate a change in subcellular GABA_AR distribution pattern. For example, BDNF may promote internalization of GABA_AR reducing high-intensity immunofluorescence associated with GABA_AR concentrated on the cell membrane, while increasing intracellular GABA_AR immunofluorescence resulting in no change in mean fluorescence intensity within cell ROIs. While we were not able to quantify a shift in subcellular localization of GABA_AR, high-magnification images (Fig. 5, D and H) indicated a disappearance of high-intensity immunoreactive structures around BDNFmyc-expressing neurons, while diffuse, lower intensity intracellular GABA_AR immunofluorescence remained present. Similar BDNF-mediated sequestration of membrane GABA_AR-α1 subunits were documented previously both in the hippocampus and amygdala (95). These changes in ROI-based GABA_AR immunofluorescence together with the significant decrease in overall GABA_AR immunofluorescence measured within the whole PVN clearly suggests that GABA_AR-mediated cardiovascular regulation in the PVN is diminished at least in part due to BDNF-mediated inhibitory action on GABA_AR expression.

In addition to directly inhibiting GABA_AR expression and signaling, BDNF can also disrupt GABAergic neurotransmission by reducing Cl⁻ gradient across the cell membrane. BDNF has been shown to decrease both mRNA and protein expression of KCC2, a neuron-specific K⁺/Cl⁻ cotransporter, which maintains low intracellular Cl⁻ concentration essential for hyperpolarizing GABA_AR-mediated currents (44, 45, 47, 48). Importantly, Choe et al. (96) have shown that a similar, BDNF-mediated KCC2 downregulation also occurs in hypothalamic vasopressin neurons and plays an important role in high salt intake-induced blood pressure elevation. Our results support these previous findings, since we found that KCC2 immunofluorescence was significantly lowered by BDNF overexpression both in ROI-based analysis and in the whole PVN. However, previous evidence suggest that it is also possible that these BDNF-mediated effects on KCC2 are secondary to augmented glutamate signaling induced by BDNF. For example, increased NMDAR activity was shown to downregulate KCC2 expression resulting in diminished Cl⁻ gradient and depolarizing GABA_AR currents in dissociated rat neurons (97). On the other hand, these actions may be countered or compensated by glutamate/mGluR signaling, which was shown to elevate KCC2 activity via protein kinase C signaling (98, 99).

In summary, this study extends our previous findings on hypertensive actions of BDNF signaling within the PVN and demonstrates that BDNF shifts the balance between excitatory and inhibitory input received by PVN neurons by upregulating NMDAR immunoreactivity and augmenting NMDA-mediated neurotransmission and downregulating GABA_AR and KCC2 immunoreactivity and diminishing GABA_AR-mediated neurotransmission. These BDNF-mediated mechanisms may synergistically contribute to elevate PVN presympathetic neuronal activity, sympathetic tone and blood pressure, and may play a significant role in the development of hypertension and related cardiovascular diseases.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grant R01 HL133211 to B. Erdos.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.T. and B.E. conceived and designed research; D.T., Z.E. and B.E. performed experiments; D.T. and B.E. analyzed data; D.T. and B.E. interpreted results of experiments; D.T. and B.E. prepared figures; D.T. and B.E. drafted manuscript; D.T., Z.E. and B.E. edited and revised manuscript; D.T., Z.E. and B.E. approved final version of manuscript.