Corticotropin-releasing hormone neurons in the central nucleus of amygdala are required for chronic stress-induced hypertension

Abstract

Aims: Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension.

Methods and results: Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs.

Conclusions: CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.

Keywords: Central nucleus of the amygdala; Chronic stress; Corticotropin-releasing hormone; Hypertension; Kv7 channel.

Graphical Abstract

Figure 1

Chronic unpredictable stress (CUS) induced a sustained elevation of blood pressure and a long-lasting hyperactivity of CeA-CRH neurons in BHRs. (A and B) Mean ABP (A) and HR (B) were monitored by radiotelemetry before, during, and after CUS in WKY rats (n = 6) and BHRs (n = 7) and age-matched unstressed BHRs (n = 7). Two-way ANOVA with post hoc analyses of Tukey multiple comparison tests were used to compare daily mean ABP and HR between experimental groups. (C) Schematic diagram depicts constructs of AAV vector containing an eGFP sequence driven by the rat CRH promoter. Inset: An eGFP-tagged CeA neuron indicated by * with a recording electrode indicated by ^ under transmitted light and fluorescent illuminant. (D) Diagram and microphotography show the eGFP expression in the CeA region. (E) Immunofluorescence images depict that eGFP-tagged neurons were positive for CRH immunoreactivity. (F) Original traces and summary data show that CUS induced a long-lasting increase in the spontaneous firing activity of CeA-CRH neurons in BHRs (n = 7 neurons from three rats in groups of Day 7 and Day 14 post-CUS) compared with unstressed BHRs (n = 6 neurons from three rats). *P < 0.05, compared with the basal values in CUS-BHRs; #P < 0.05, compared with respective values in unstressed BHRs; repeated-measures 2-way ANOVA with Dunnett’s post hoc test. OT, optic tract.

Figure 2

Chemogenetic inhibition of CeA-CRH neurons effectively alleviated CUS-induced sustained hypertension in BHRs. (A) Schematic diagram shows the structures of viral vectors, injection of the viral vectors, and expression of hM4Di on CeA-CRH neurons. Inset images show a mCherry-tagged CeA neuron viewed under transmitted light and fluorescent illuminant. (B) Immunofluorescence images show the mCherry-tagged CeA neurons were positive for CRH immunoreactivity. (C and D) Representative raw tracings (C) and summary data (D) depict that bath application of 10 μM C21 selectively decreased the firing activity of mCherry-tagged CRH neurons (n = 6 neurons from three unstressed BHRs) but did not alter the firing activity of unlabelled neurons (n = 6 neurons from three BHRs). Repeated-measures ANOVA with Dunnett’s post hoc test was used to compare the firing activity between groups. (E and F) CUS elevated mean ABP (E) and HR (F) in BHRs injected with AAV-DIO-hM4Di viral vector (n = 7) or co-injection of AAV-hSyn-DIO-mCherry and AAV-CRH-Cre into the CeA vectors. Administration of C21 (1.0 mg/kg/day for 28 days) through implanted osmotic pump significantly mitigated CUS-induced pressor response and tachycardia in BHRs injected with AAV-CRH-Cre and AAV-DIO-hM4Di viral vectors (n = 8). Two-way ANOVA with post hoc analyses of Tukey multiple comparison tests were used to compare daily mean ABP and HR between experimental groups. *P < 0.05, compared with baseline values; #P < 0.05, compared with BHRs injected with AAV-DIO-hM4Di.

Figure 3

Chemogenetic inhibition of CeA-CRH neurons effectively decreased sympathetic outflow and ABP in CUS-treated BHRs. (A and B) Raw recording traces show that intravenous bolus injection of C21 (1.0 mg/kg) decreased ABP, RSNA, and HR in CUS-treated BHRs injected with AAV-CRH-Cre and AAV-DIO-hM4Di into the CeA (A), while C21 did not alter ABP, RSNA, and HR in BHRs injected with AAV-DIO-hM4Di into the CeA (B). Recording of ABP, RSNA, and HR in CUS-BHRs were performed 14 days after termination of CUS treatment. (C, D, and E) Summary data show changes in mean ABP (C), RSNA (D), and HR (E) in response to C21 (i.v. 1.0 mg/kg) in six CUS-BHRs injected with AAV-CRH-Cre and AAV-DIO-hM4Di and BHRs injected with only AAV-DIO-hM4Di (n = 6). *P < 0.05, compared with the respective baseline. Repeated-measures ANOVA with Dunnett’s post hoc test.

Figure 4

CUS decreased protein and mRNA expression levels of Kv7.2, Kv7.3, and Kv7.5. (A, B, and C) Original gel images (A), quantification of immunoblot band density (B), and summary data of mRNA levels (C) show the total protein and mRNA levels of Kv7.2, Kv7.3, and Kv7.5 in the CeA in unstressed and CUS-treated BHRs (n = 6 samples in each group). (D, E, and F) Original gel images (D), quantification of band density (E), and summary data of mRNA levels (F) show the protein and mRNA levels (n = 6 samples in each group) of Kv7.2, Kv7.3, and Kv7.5 in the PVN in unstressed and CUS-treated BHRs. In these protein assays, each sample consists of respective tissues from one rat. *P < 0.05, compared with values in unstressed BHRs (unpaired Student’s t-test). PVN, paraventricular nucleus.

Figure 5

CUS decreases M-currents in CeA-CRH neurons and the excitatory effect of Kv7 channel blocker. (A) Immunofluorescence images show the distribution of Kv7.2, Kv7.3, and Kv7.5 immunoreactivities on CeA-CRH neurons. (B) Raw traces of voltage-clamp recording show M-currents in CeA-CRH neurons before and after applying an M-channel blocker XE-991 (10 μM) and an M-channel opener retigabine (10 μM) in unstressed BHRs and CUS-treated BHRs at Day 7 and Day 14 after the termination of CUS. (C) Summary data shows that calculated basal and total M-currents were significantly diminished in CeA-CRH neurons in CUS-treated BHRs after the termination of CUS (n = 9 neurons in four CUS-treated BHRs at Day 7 post-CUS and n = 8 neurons in four CUS-treated BHRs at Day 14 post-CUS) compared with unstressed BHRs (n = 7 neurons in four BHRs). *P < 0.05, compared with basal M current density in unstressed BHRs; #P < 0.05, compared with total M current density in unstressed BHRs. (D and E) Raw tracings (D) and summary data (E) show the effect of Kv7 channel blocker XE-991 (10 µM) on the spontaneous firing activity of CeA-CRH neurons in unstressed BHRs (n = 7 neurons from four BHRs), CUS-treated BHRs at Days 7 and 14 post-CUS (n = 8 neurons from four BHRs in each group). *P < 0.05, compared with the baseline values in the group; #P < 0.05, compared with the corresponding values in unstressed BHRs. Repeated-measures ANOVA with Dunnett’s post hoc test. RB, retigabine.

Figure 6

Blocking Kv7 channels in the CeA increased sympathetic output in unstressed BHRs but not in CUS-treated BHRs. (A) Diagram and representative microphotography (upper panel) and schematic drawings (lower panels) depict the microinjection sites of XE-991 in the CeA in unstressed BHRs and CUS-treated BHRs. (B and C) Raw recording traces show the effect of microinjection of XE-991 into the CeA on ABP, RSNA, and HR in unstressed BHRs (B) and CUS-treated BHRs (C). (D, E, and F) Summary data show changes in mean ABP (D), RSNA (E), and HR (F) in response to microinjection of XE-991 into the CeA in unstressed BHRs (n = 7) and CUS-treated BHRs (n = 6). *P < 0.05, ***P < 0.0001, compared with the respective baseline values; #P < 0.05, compared with basal values in unstressed BHRs. Repeated-measures ANOVA with Dunnett's post hoc test. 3V, third ventricle.