An Angiotensin-Responsive Connection from the Lamina Terminalis to the Paraventricular Nucleus of the Hypothalamus Evokes Vasopressin Secretion to Increase Blood Pressure in Mice

Abstract

Blood pressure is controlled by endocrine, autonomic, and behavioral responses that maintain blood volume and perfusion pressure at levels optimal for survival. Although it is clear that central angiotensin type 1a receptors (AT1aR; encoded by the Agtr1a gene) influence these processes, the neuronal circuits mediating these effects are incompletely understood. The present studies characterize the structure and function of AT1aR neurons in the lamina terminalis (containing the median preoptic nucleus and organum vasculosum of the lamina terminalis), thereby evaluating their roles in blood pressure control. Using male Agtr1a-Cre mice, neuroanatomical studies reveal that AT1aR neurons in the area are largely glutamatergic and send projections to the paraventricular nucleus of the hypothalamus (PVN) that appear to synapse onto vasopressin-synthesizing neurons. To evaluate the functionality of these lamina terminalis AT1aR neurons, we virally delivered light-sensitive opsins and then optogenetically excited or inhibited the neurons while evaluating cardiovascular parameters or fluid intake. Optogenetic excitation robustly elevated blood pressure, water intake, and sodium intake, while optogenetic inhibition produced the opposite effects. Intriguingly, optogenetic excitation of these AT1aR neurons of the lamina terminalis also resulted in Fos induction in vasopressin neurons within the PVN and supraoptic nucleus. Further, within the PVN, selective optogenetic stimulation of afferents that arise from these lamina terminalis AT1aR neurons induced glutamate release onto magnocellular neurons and was sufficient to increase blood pressure. These cardiovascular effects were attenuated by systemic pretreatment with a vasopressin-1a-receptor antagonist. Collectively, these data indicate that excitation of lamina terminalis AT1aR neurons induces neuroendocrine and behavioral responses that increase blood pressure.SIGNIFICANCE STATEMENT Hypertension is a widespread health problem and risk factor for cardiovascular disease. Although treatments exist, a substantial percentage of patients suffer from "drug-resistant" hypertension, a condition associated with increased activation of brain angiotensin receptors, enhanced sympathetic nervous system activity, and elevated vasopressin levels. The present study highlights a role for angiotensin Type 1a receptor expressing neurons located within the lamina terminalis in regulating endocrine and behavioral responses that are involved in maintaining cardiovascular homeostasis. More specifically, data presented here reveal functional excitatory connections between angiotensin-sensitive neurons in the lamina terminals and vasopressin neurons in the paraventricular nucleus of the hypothalamus, and further indicate that activation of this circuit raises blood pressure. These neurons may be a promising target for antihypertensive therapeutics.

Keywords: angiotensin Type 1 receptors; blood pressure; hypertension; median preoptic nucleus; renin-angiotensin system.

Figure 1.

AT1aR neurons within the MnPO and OVLT are predominantly glutamatergic. Images of the (A–D) MnPO and (E–H) OVLT collected from an Agtr1a-tdTomato (AT1aR-Tom) reporter mouse depicting (B,F) vGlut2 mRNA in cyan, (C,G) AT1aR-tdTom in magenta, and (A,D,E,H) the merged images. I–L, Images of the MnPO collected from an AT1aR-Tom reporter mouse depicting (J) Gad1 mRNA in green, (K) AT1aR-Tom in magenta, and (L) the merged image. n = 4 mice. Scale bars: A, E, I, 50 µm; B–D, F–H, J–L, 20 µm.

Figure 2.

Angiotensin-II excites neurons in the MnPO/OVLT, and this response is attenuated by antagonism of AT1aR. A, AAV delivery of Cre-dependent GCaMP6f into the MnPO/OVLT of Agtr1a-Cre mice mouse selectively transforms AT1aR neurons. B, Exposure to Ang-II (1 μm, 3 min) increases fluorescence in GGaMP6f-expressing neurons. C, The excitatory action of Ang-II (n = 6, blue trace) is attenuated by pretreatment with Losartan (10 μm, n = 4, red trace). aca, anterior commissure. Scale bars, 250 µm.

Figure 3.

AT1aR neurons of the MnPO/OVLT send projections to brain regions involved in body fluid homeostasis and blood pressure regulation. A, Schematic illustrating (Ai) the viral construct used to direct the Cre-dependent expression of ChR2 and eYFP to neurons within the MnPO/OVLT of the Agtr1a-Cre mouse; (Aii) the injection of the AAV into the MnPO/OVLT; and (Aiii) the subsequent Cre-mediated inversion of the dual floxed eYFP and ChR2 into the correct orientation. B, Coronal section through the OVLT and MnPO depicting Cre-dependent expression of eYFP (green) in the somas of neurons expressing AT1aR and their fibers in the BNST (dashed box). Coronal section through the (C) SFO demonstrating eYFP-labeled axons originating from AT1aR neurons of the MnPO/OVLT. D, E, Unilateral coronal section through the PVN illustrating that axons (green) arising from neurons in the MnPO/OVLT that express AT1aR make appositions onto AVP (magenta) synthesizing neurons. Coronal section through the (F) DMH demonstrating eYFP-labeled axons originating from AT1aR neurons of the MnPO/OVLT. 3v, Third cerebral ventricle. Images are representative of 8 mice. Scale bars: B, 200 µm; C, D, 50 µm; E, 5 µm; F, 100 µm.

Figure 4.

Optogenetic stimulation of AT1aR-containing neurons in the MnPO/OVLT influences blood pressure. A, Cre-dependent expression of ChR2/eYFP (green) in MnPO/OVLT of an Agtr1a-Cre mouse. B, A combination of epifluorescence and DIC microscopy was used to target virally transformed neurons for study. C, Exposure to 15 Hz blue light produces action potentials. D, SBP and (E) heart rate (HR) response to blue light stimulation of AT1aR neurons residing in the MnPO/OVLT (10 mW; 15 Hz; 20 ms pulse width; 60 s ON/OFF; performed over a period of 10 min, n = 4 or 5/group). Water and 0.3 m NaCl intake in response to the same stimulation parameters (F) during free access to water and (G) subsequent to 16 h of water deprivation; n = 15 mice. H, Cre-dependent expression of halorhodopsin (Halo)/eYFP (green) in the MnPO/OVLT of an AT1aR-Cre mouse. I, A combination of epifluorescence and DIC microscopy was used to target virally transformed neurons for study. J, Green light hyperpolarizes AT1aR neurons in the MnPO/OVLT. Changes in (K) SBP and (L) HR in response to optogenetic inhibition of AT1aR neurons of the MnPO/OVLT in mice receiving AAV-Halo; n = 6/group. Water and 0.3 m NaCl intake in response to optogenetic inhibition (M) during free access to water and (N) subsequent to 16 h of water deprivation; n = 9 mice. aca, anterior commissure. Error bars indicate SEM. *p < 0.05. Scale bars: A, H, 250 µm; B, I, 20 µm.

Figure 5.

Stimulation of AT1aR-containing neurons within the MnPO/OVLT activates neurons in the PVN and SON that synthesize AVP. Control mice (eYFP) and mice receiving the Cre-dependent AAV for the expression the ChR2 and eYFP were subjected to the same optogenetic stimulation protocol as in Figure 4. Ninety minutes after the onset of optogenetic stimulation, mice were killed and perfused, and brains were processed for Fos immunoreactivity. Representative projection images through the OVLT and MnPO of (A,C) an eYFP control and (B,D) a ChR2 mouse subjected to optogenetic stimulation. Magenta represents Fos. Green represents AT1aR-expressing neurons. Representative projection images through the SON and PVN of an (E,G) eYFP control and a (F,H- J) ChR2 mouse depicting AT1aR-eYFP neuronal fibers (green) arising from the MnPO/OVLT, AVP (magenta), and Fos immunoreactivity (cyan). K, Total Fos-positive nuclei/section in the brain ROIs and (L) the percentage of AVP neurons containing Fos within the SON and PVN. Error bars indicate SEM. n = 7/group. *p < 0.05. Scale bars: A–F, 100 µm; G, H, 50 µm; I, J, 5 µm.

Figure 6.

Optogenetic stimulation of axons in the PVN that originate from MnPO/OVLT AT1aR neurons releases glutamate (GLU) onto magnocellular (MCN) PVN neurons and induces a V1aR-dependent elevation in blood pressure. A, Experimental design for in vitro optogenetic study. B, PVN magnocellular neurons were initially targeted based on their location, large soma, and general morphology as apparent under IR-DIC. C, Classification as a PVN magnocellular neuron was confirmed during current-clamp recordings based on the presence of a large I_A current. This current is responsible for the long delay to first action potential observed in response to suprathreshold stimulation following a brief hyperpolarizing step (arrow). D, Blue light stimulation (BLS) evoked EPSCs in 15 of 65 magnocellular neurons tested. A subset of light-evoked responses observed (5 of 15) were challenged with ionotropic glutamate receptor antagonists (DNQX and AP5), which effectively eliminated the response (inset). E, Post hoc immunohistochemical assessment of a light-responsive PVN magnocellular neuron suggests an AVP phenotype. F, Schematic depicting the experimental design used to determine the role of V1aR in cardiovascular responses to blue light stimulation of afferents arising from AT1aR-containing neurons of the MnPO/OVLT. For these studies, the AAV-ChR2 or AAV-eYFP was injected into the MnPO/OVLT of Agtr1a-Cre mice and the fiber-optic post was positioned over the PVN. G, SBP (top) and HR (bottom) response to blue light stimulation of axons in the PVN arising from MnPO/OVLT AT1aR-expressing neurons (10 mW; 15 Hz; 60 s). n = 4 or 5/group. H, Illustration of the overall conclusion that AT1aR neurons in the MnPO/OVLT elicits elevations in blood pressure, in part, via excitatory connections to neurons in the PVN that secrete AVP into the systemic circulation. Error bars indicate SEM. *p < 0.05. Scale bars, 20 µm.