Cardiovascular (CV) representation has been identified within the insular cortex (IC) and a lateralization of function previously suggested. In order to further understand the role of IC on cardiovascular control, the present study compared the CV responses evoked by stimulation of N-metil-D-aspartate (NMDA) receptors in the right and left posterior IC at different rostrocaudal levels. Intracortical microinjections of NMDA were performed into the IC of male Wistar rats anaesthetized with urethane (1.4 g/kg) prepared for blood pressure, heart rate and renal sympathetic nerve activity. Gene expression of NMDA receptor subunits NR2A and NR2B in the IC was confirmed by RT-PCR. Immunofluorescence for the NMDA receptor NR1 subunit was demonstrated in the IC (coordinates anteroposterior (AP) +1.5, 0.0 and -1.5 mm). A cardiac sympathoinhibitory site was identified, more rostrally located than identified in previous studies. A site of sympathoexcitatory cardiac control was identified more caudal to this region in agreement with earlier work. Under the experimental conditions, no lateralization of cardiovascular function was identified with chemical stimulation eliciting the same responses from either left or right insular cortices. No tonic role of the insula on cardiovascular control was identified with the use of the NMDA antagonist, AP-5. Peri-insular microinjection of NMDA was without cardiovascular effect indicating the specificity of the insula as a cardiovascular regulatory site. The current study reveals a functional topography for autonomic cardiovascular control along the rostrocaudal axis of the posterior IC.

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Experiments aimed to evaluate the tissue distribution of Mas-related G-protein coupled receptor D (MrgD) revealed the presence of immunoreactivity for the MrgD protein in the rostral insular cortex (rIC), an important area for autonomic and cardiovascular control. In order to investigate the relevance of this finding, we evaluated the cardiovascular effects produced by the endogenous ligand of MrgD, alamandine, in this brain region. Mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were recorded in urethane anesthetized rats. Unilateral microinjection of equimolar doses of alamandine (40pmol/100nl), angiotensin-(1-7), angiotensin II, angiotensin A and Mas/MrgD antagonist D-Pro7-Ang-1-7 (50pmol/100nl), Mas antagonist A779 (100pmol/100nl) or vehicle (0.9% NaCl) were made in different rats (N=4-6 per group) into rIC. To verify the specificity of the region, a microinjection of alamandine was also performed into intermediate insular cortex (iIC). Microinjection of alamandine in rIC produced an increase in MAP (Δ=15±2mmHg), HR (Δ=36±4bpm) and RSNA (Δ=31±4%), but was without effects at iIC. Strikingly, an equimolar dose of angiotensin-(1-7) at rIC did not produce any change in MAP, HR and RSNA. Angiotensin II and angiotensin A produced only minor effects. Alamandine effects were not altered by A-779, a Mas antagonist, but were completely blocked by the Mas/MrgD antagonist D-Pro7-Ang-(1-7). Therefore, we have identified a brain region in which alamandine/MrgD receptor but not angiotensin-(1-7)/Mas could be involved in the modulation of cardiovascular-related neuronal activity. This observation also suggests that alamandine might possess unique effects unrelated to angiotensin-(1-7) in the brain.

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The intermediate region of the posterior insular cortex (intermediate IC) mediates sympathoexcitatory responses to the heart and kidneys. Previous studies support hypertension‐evoked changes to the structure and function of neurons, blood vessels, astrocytes and microglia, disrupting the organization of the neurovascular unit (NVU). In this study, we evaluated the functional and anatomical integrity of the NVU at the intermediate IC in the spontaneously hypertensive rat (SHR) and its control the Wistar‐Kyoto (WKY). Under urethane anesthesia, NMDA microinjection (0.2 mmol/L/100 nL) was performed at the intermediate IC with simultaneous recording of renal sympathetic nerve activity (RSNA), heart rate (HR) and mean arterial pressure (MAP). Alterations in NVU structure were investigated by immunofluorescence for NMDA receptors (NR1), blood vessels (70 kDa FITC‐dextran), astrocytes (GFAP), and microglia (Iba1). Injections of NMDA into intermediate IC of SHR evoked higher amplitude responses of RSNA, MAP, and HR. On the other hand, NMDA receptor blockade decreased baseline RSNA, MAP and HR in SHR, with no changes in WKY. Immunofluorescence data from SHR intermediate IC showed increased NMDA receptor density, contributing to the SHR enhanced sympathetic responses, and increased in vascular density (increased number of branches and endpoints, reduced average branch length), suggesting angiogenesis. Additionally, IC from SHR presented increased GFAP immunoreactivity and contact between astrocyte processes and blood vessels. In SHR, IC microglia skeleton analysis supports their activation (reduced number of branches, junctions, endpoints and process length), suggesting an inflammatory process in this region. These findings indicate that neurogenic hypertension in SHR is accompanied by marked alterations to the NVU within the IC and enhanced NMDA‐mediated sympathoexcitatory responses likely contributors of the maintenance of hypertension.

Angiotensin II (Ang II) acts as a pro-stress hormone, while other evidence indicates that angiotensin-(1-7) [Ang-(1-7)] attenuates physiological responses to emotional stress. To further test this hypothesis, in groups of 5-6 rats we evaluated autonomic, cardiovascular and behavioral parameters in male Sprague-Dawley (SD) and transgenic TGR(A1-7)3292 (TG) rats chronically overexpressing Ang-(1-7). Compared to SD rats, TG rats showed reduced baseline heart rate (HR; SD 380 ± 16 versus TG 329 ± 9 beats per minute (bpm), mean ± standard error of mean, p< .05) and renal sympathetic discharge (SD 138 ± 4 versus TG 117 ± 5 spikes/second, p < .05). TG rats had an attenuated tachycardic response to acute air-puff stress (ΔHR: SD 51 ± 20 versus TG 1 ± 3 bpm; p < .05), which was reversed by intracerebroventricular injection of the Mas receptor antagonist, A-779 (ΔHR: SD 51 ± 20 versus TG 63 ± 15 bpm). TG rats showed less anxious behavior on the elevated plus maze, as revealed by more entries into open arms (SD 2 ± 2 versus TG 47 ± 5% relative to total entries; p < .05), and more time spent in the open arms (SD 5 ± 4 versus TG 53 ± 9% relative to total time, p < .05). By contrast with SD rats, diazepam (1.5 mg/kg, intraperitoneally) did not further reduce anxious behavior in TG rats, indicating a ceiling anxiolytic effect of Ang-(1-7) overexpression. Ang-(1-7) concentrations in hypothalamus and plasma, measured by mass spectrometry were two- and three-fold greater, respectively, in TG rats than in SD rats. Hence, increased endogenous Ang-(1-7) levels in TG rats diminishes renal sympathetic outflow and attenuates cardiac reactivity to emotional stress, which may be via central Mas receptors, and reduces anxious behavior. Lay summaryWe used a genetically modified rat model that produces above normal amounts of a peptide hormone called angiotensin-(1-7) to test whether this peptide can reduce some of the effects of stress. We found that angiotensin-(1-7), acting in the brain, can reduce anxiety and reduce the increase in heart rate associated with emotional stress. These findings may provide a lead for design of new drugs to reduce stress.

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Thus, an avenue of knowledge may unfold from a field of research that requires further exploration.