Blood-borne angiotensin II acts in the brain to influence behavioral and endocrine responses to psychogenic stress

Abstract

This study elucidates the neural circuits by which circulating angiotensin II (ANGII) acts in the brain to influence humoral and behavioral responses to psychological stressors. To test the hypothesis that systemic ANGII mediates stress responding via the subfornical organ (SFO), we first found that the timing of increased systemic ANGII in response to 60 min restraint coincides with increased c-fos mRNA expression in the SFO. Next, we administered an anterograde neuronal tract tracer into the SFO and found that fibers originating there make appositions onto neurons in the paraventricular nucleus of the hypothalamus that are also c-fos positive following restraint stress. To determine whether circulating ANGII stimulates the release of stress hormones via activation of angiotensin type 1 receptors (AT1R) within the SFO, we delivered lentivirus to knockdown AT1R expression locally in the SFO. Inhibition of AT1R specifically within the SFO blunted the release of adrenocorticotrophin-releasing hormone and corticosterone in response to restraint stress and caused rats to spend more time in the open arms of an elevated-plus maze than controls, indicating that inhibition of AT1R within the SFO is anxiolytic. Collectively, these results suggest that circulating ANGII acts on AT1R in the SFO to influence responding to psychological stressors.

Conflict of interest statement

The authors have no conflicts of interest.

Figures

Figure 1.

a, Sixty-minute restraint elicits elevations in ACTH, CORT, and PRA with similar peaks at 15 min (n = 8 per group and time point). b, Restraint significantly increases c-fos mRNA in the SFO [asterisk (*), different than 0 min; pound sign (#), different than 120 min; *,#p < 0.05, n = 8 per group). c, Restraint-induced c-fos mRNA expression in the SFO is attenuated by systemic pretreatment with losartan (*p < 0.05, n = 6 per group).

Figure 2.

a, Drawing of coronal section through the SFO highlighted in red. b–d, Representative cases of rats administered PHA-L into the SFO (green, Alexa Fluor 488, PHA-L-positive fibers). Scale bars, 250 μm).

Figure 3.

(a) High-magnification image of a neuron in the anterior parvocellular PVN that is activated during restraint and receives direct efferent projections from the SFO; efferent fibers result from injection site shown in Figure 2c. Scale bar, 25 μm. b, Lower-magnification image of neurons in the medial parvocellular PVN that are activated during restraint and receive direct projections from the SFO; efferent fibers result from injection site shown in Figure 2d. Scale bar, 50 μm. c, High-magnification image of neurons in the anterior/medial parvocellular PVN that are activated during restraint and receive direct projections from the SFO; efferent fibers result from injection site shown in Figure 2b. Scale bar, 25 μm. d, Lower-magnification image of neurons in the medial parvocellular PVN that are activated during restraint and receive direct projections from the SFO; efferent fibers result from injection site shown in Figure 2d. Scale bar, 50 μm. Insets, Drawings of unilateral coronal sections through the anterior or medial parvocellular PVN depicting the location of the triple labeled neurons (red, Cy3-positive label for HuCD; blue, Cy5 c-fos-positive nuclei; green, Alexa Fluor 488 PHA-L fibers from SFO).

Figure 4.

a, Representative coronal sections through the SFO from rats administered lentivirus. Top left, Neurons within the SFO express GFP. Top right, Adjacent section stained for NeuN. Scale bar, 250 μm. Bottom left, Autoradiograph indicative of ANGII binding in the SFO of rats administered LV-GFP. Bottom right, Autoradiograph indicative of ANGII binding in the SFO of rats administered LV-AS. Scale bar, 1 mm. b, Receptor autoradiography indicative of ANGII binding in the SFO and PVN. Administration of LV-AS significantly decreases (*p < 0.05) ANGII binding in the SFO but has no effect on the PVN (LV-GFP SFO, n = 8; LV-AS SFO, n = 8; LV-GFP PVN, n = 5; LV-AS PVN, n = 5).

Figure 5.

a, b, Administration of LV-AS into the SFO significantly decreases (*p < 0.05) the release of ACTH (a) and CORT (b) during restraint (ACTH LV-GFP, n = 12; ACTH LV-AS, n = 12; CORT LV-GFP, n = 16; CORT LV-AS, n = 15). c, d, Delivery of LV-AS into the SFO significantly increases (*p < 0.05) the amount of time spent in the open arms of the EPM but has no effect on total distance traveled (n = 12 per group).