Forebrain Circumventricular Organs Research Articles

Abstract 54: The kidney-OVLT axis in renovascular hypertension: Individual and combined effects of afferent renal denervation and OVLT lesion in 2K1C rats.

We have recently reported that afferent renal denervation (ARDN) markedly attenuates the pathogenesis of hypertension in the 2 kidney 1 clip (2K1C) model in the rat. This was associated with a reduction in neurogenic pressor activity (NPA). We have also recently shown that lesion of the organum vasculosum of the lamina terminalis (OVLT), a forebrain circumventricular organ, had nearly identical effects in 2K1C rats as ARDN. Together these findings suggest that a neural pathway from the kidney to the OVLT plays an important role in 2K1C hypertension. Based on these findings we hypothesized that the combined effect of ARDN and lesion of the OVLT (OVLTx) on arterial pressure in 2K1C rats would not be greater than the individual treatments. Male Sprague-Dawley rats were randomly selected to receive either OVLT lesion (x) or sham-OVLTx. One week later, rats were instrumented with telemeters to measure mean arterial pressure (MAP). One week later, renal artery stenosis was created by placing a clip on the left renal artery and then, rats were subjected to ARDN by periaxonal capsaicin or sham treatment of the clipped kidney creating 4 experimental groups: sham+sham (n=6), sham+ARDN (n=6), OVLTx+sham (n=5), OVLTx+ARDN (n=5). MAP was measured daily for 28 days, and at the end of the protocol, NPA was assessed by measuring the peak MAP response to ganglionic blockade. MAP was similar in all 4 groups at the beginning of the study averaging 108±3 mmHg. MAP increased to 180±10 mmHg in the sham+sham group compared to 130±13mmHg in sham+ARDN, 142±18mmHg in OVLTx+sham rats and 158±14mmHg in OVLTx+ARDN rats (p<0.001). NPA in Sham+sham rats was significantly higher (-44 ± 8 mmHg) compared to sham+ARDN (-18 ± 3 mmHg), OVLTx+sham (-21 ± 2 mmHg) and OVLTx+ARDN (-19 ± 4 mmHg) rats (p<0.05). Notably, the combined effects of OVLTx+ARDN on MAP and NPA were not significantly different from either OVLTx (p=1.00 and p=0.819) or ARDN (p=0.113 and p= 0.878) alone. In summary, ARDN and OVLTx attenuated hypertension and NPA similarly in 2K1C-HTN rats but the combination of these treatments did not produce any additional effect on MAP or NPA. These findings are consistent with the hypothesis that afferent renal nerves and the OVLT are linked in a common neural pathway required for the development of hypertension following renal artery stenosis.

AT1 receptors in the subfornical organ modulate arterial pressure and the baroreflex in two-kidney, one-clip hypertensive rats.

The subfornical organ (SFO), a forebrain circumventricular organ that lies outside the blood-brain barrier, has been implicated in arterial pressure and baroreflex responses to angiotensin II (ANG II). We tested whether pharmacological inhibition or selective silencing of SFO ANG II type 1 receptors (AT1R) of two-kidney, one-clip rats with elevated plasma ANG II decreases resting arterial pressure and renal sympathetic nerve activity (RSNA) and/or modulates arterial baroreflex responses of heart rate (HR) and RSNA. Male Sprague-Dawley rats underwent renal artery clipping [2-kidney, 1-clip (2K,1C)] or sham clipping (sham). After 6 wk, conscious rats instrumented with vascular catheters, renal nerve electrodes, and a cannula directed to the SFO were studied. In another set of experiments, rats were instrumented with hemodynamic and nerve radio transmitters and injected with scrambled RNA or silencing RNA targeted against AT1R. Mean arterial pressure (MAP) was significantly higher in 2K,1C rats. Acute SFO injection with the AT1R inhibitor losartan did not change MAP in sham or 2K,1C rats. Baroreflex curves of HR and RSNA were shifted rightward in 2K,1C rats. Losartan exerted no effect. SFO AT1R knockdown did not influence MAP in sham rats but decreased MAP in 2K,1C rats, despite no change in plasma ANG II or resting RSNA. AT1R knockdown prevented the reduction in maximum gain and slope of baroreflex responses of HR and RSNA; the reduced RSNA response to baroreceptor unloading was partially restored in 2K,1C rats. These findings show that AT1R activation within the SFO contributes to hypertension and baroreflex dysfunction in 2K,1C rats and highlight the temporal requirement for reversal of these effects.

TNF-α receptor 1 knockdown in the subfornical organ ameliorates sympathetic excitation and cardiac hemodynamics in heart failure rats.

In systolic heart failure (HF), circulating proinflammatory cytokines upregulate inflammation and renin-angiotensin system (RAS) activity in cardiovascular regions of the brain, contributing to sympathetic excitation and cardiac dysfunction. Important among these is the subfornical organ (SFO), a forebrain circumventricular organ that lacks an effective blood-brain barrier and senses circulating humors. We hypothesized that the tumor necrosis factor-α (TNF-α) receptor 1 (TNFR1) in the SFO contributes to sympathetic excitation and cardiac dysfunction in HF rats. Rats received SFO microinjections of a TNFR1 shRNA or a scrambled shRNA lentiviral vector carrying green fluorescent protein, or vehicle. One week later, some rats were euthanized to confirm the accuracy of the SFO microinjections and the transfection potential of the lentiviral vector. Other rats underwent coronary artery ligation (CL) to induce HF or a sham operation. Four weeks after CL, vehicle- and scrambled shRNA-treated HF rats had significant increases in TNFR1 mRNA and protein, NF-κB activity, and mRNA for inflammatory mediators, RAS components and c-Fos protein in the SFO and downstream in the hypothalamic paraventricular nucleus, along with increased plasma norepinephrine levels and impaired cardiac function, compared with vehicle-treated sham-operated rats. In HF rats treated with TNFR1 shRNA, TNFR1 was reduced in the SFO but not paraventricular nucleus, and the central and peripheral manifestations of HF were ameliorated. In sham-operated rats treated with TNFR1 shRNA, TNFR1 expression was also reduced in the SFO but there were no other effects. These results suggest a key role for TNFR1 in the SFO in the pathophysiology of systolic HF.NEW & NOTEWORTHY Activation of TNF-α receptor 1 in the subfornical organ (SFO) contributes to sympathetic excitation in heart failure rats by increasing inflammation and renin-angiotensin system activity in the SFO and downstream in the hypothalamic paraventricular nucleus. Cytokine receptors in the SFO may be a target for central intervention in cardiovascular conditions characterized by peripheral inflammation.

Angiotensin II Type 1a Receptor Knockdown in Subfornical Organ Attenuates Central Expression of Inflammatory Mediators and Ameliorates Peripheral Manifestations of Heart Failure in Rats After Myocardial Infarction

Myocardial infarction (MI)‐induced heart failure (HF) is characterized by increased proinflammatory cytokines (PICs) and renin‐angiotensin system (RAS) activity in the periphery and in the brain, contributing to sympathetic excitation and the progression of HF. Although circulating angiotensin II (ANGII) and PICs can both up‐regulate inflammation and RAS activity in cardiovascular regions of the brain, their interactions within the brain in HF remains unclear. Receptors for ANGII and PICs are highly expressed in the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood–brain barrier and senses circulating humoral factors. We previously reported (FASEB J., 30:1b719, 2016) that knockdown of the tumor necrosis factor‐α (TNF‐α) receptor 1 (TNFR1) in the SFO of HF rats reduced the expression of RAS and inflammatory components in the SFO and downstream in the hypothalamic paraventricular nucleus (PVN), with improvements in indices of sympathetic drive and hemodynamic function. The present study examined the effect of knocking down the ANGII type 1a receptor (AT1aR) in the SFO of HF rats. Rats received SFO microinjections of an adeno‐associated viral vector carrying AT1aR shRNA or a scrambled shRNA and green fluorescent protein (GFP). One week later, transfection potential and knockdown efficiency were examined in some rats receiving shRNAs; other rats underwent coronary ligation to induce HF confirmed by echocardiography, or a sham operation. One week after SFO microinjections of AT1aR shRNA or scrambled shRNA, GFP fluorescence was visualized in the SFO but not in other brain areas. Rats receiving AT1aR shRNA also had significantly reduced AT1aR mRNA in the SFO compared with rats receiving scrambled shRNA. Four weeks after MI, HF+scrambled shRNA rats had increased mRNA for AT1aR, TNF‐α, TNFR1, interleukin (IL)‐1β, cyclooxygenase (COX)‐2, c‐fos (a marker of neuronal excitation), GFAP (an astrocyte marker) and CD68 (a microglial marker) in SFO and in PVN, along with increased plasma norepinephrine levels and lung/body weight and right ventricle/body weight ratios, compared with SHAM+scrambled shRNA rats. All of these abnormalities were ameliorated in the HF+AT1aR shRNA rats, that had reduced mRNA for AT1aR. Notably, SHAM+AT1aR shRNA rats also had reduced AT1aR mRNA in the SFO, but did not exhibit alterations in expression of the above mediators in the SFO and PVN, compared with SHAM+ scrambled shRNA rats. The results support the view that circulating ANGII, acting upon AT1aR in the SFO, upregulates the inflammatory response in the SFO with downstream effects in the PVN. Taken together with our previous study of the effects of TNFR1 knockdown in SFO, these results suggest that the SFO is a site at which reciprocal facilitatory interactions occur between RAS and PICs, both contributing to sympathetic excitation in HF, and that interrupting either may have beneficial effects. Astrocytes or microglia that express receptors for both ANGII and PICs may play a role in mediating these interactions.Support or Funding InformationSupported by NIH RO1 HL‐073986 and Department of Veterans Affairs.

Neural Control of Blood Pressure in Chronic Intermittent Hypoxia.

Sleep apnea (SA) is increasing in prevalence and is commonly comorbid with hypertension. Chronic intermittent hypoxia is used to model the arterial hypoxemia seen in SA, and through this paradigm, the mechanisms that underlie SA-induced hypertension are becoming clear. Cyclic hypoxic exposure during sleep chronically stimulates the carotid chemoreflexes, inducing sensory long-term facilitation, and drives sympathetic outflow from the hindbrain. The elevated sympathetic tone drives hypertension and renal sympathetic activity to the kidneys resulting in increased plasma renin activity and eventually angiotensin II (Ang II) peripherally. Upon waking, when respiration is normalized, the sympathetic activity does not diminish. This is partially because of adaptations leading to overactivation of the hindbrain regions controlling sympathetic outflow such as the nucleus tractus solitarius (NTS), and rostral ventrolateral medulla (RVLM). The sustained sympathetic activity is also due to enhanced synaptic signaling from the forebrain through the paraventricular nucleus (PVN). During the waking hours, when the chemoreceptors are not exposed to hypoxia, the forebrain circumventricular organs (CVOs) are stimulated by peripherally circulating Ang II from the elevated plasma renin activity. The CVOs and median preoptic nucleus chronically activate the PVN due to the Ang II signaling. All together, this leads to elevated nocturnal mean arterial pressure (MAP) as a response to hypoxemia, as well as inappropriately elevated diurnal MAP in response to maladaptations.

Estrogen Effects on Neural Activation after Short‐term Vs. Long‐term Furosemide Treatment in Rats

Estrogen influences body Na+ balance, but the central pathways involved in these effects are currently unknown. Studies using immunohistochemical labeling of the fos protein to study central areas involved in body fluid balance report no effect of Na+ loss in male rats. However, standard methods of producing Na+ loss are of comparatively long duration, and it is possible central activation is not sustained. Accordingly, our objective was to assess the effect of estrogen on activation in response to short-term and long-term Na+ depletion using furosemide, a diuretic-natriuretic. Adult female rats were ovariectomized, allowed to recover for 7 days, and then given estradiol benzoate (EB; 10 μg/0.1 ml oil, sc) or oil vehicle (OIL; 0.1 ml, sc). Rats were injected with furosemide (2 X 5 mg/kg, sc) by one of two protocols. For the short term protocol, rats were perfused with paraformaldehyde 2 hrs after furosemide; for the long-term protocol, rats were perfused 18 hrs after furosemide. Brains were removed and cut into 40μ sections and then immunolabeled for fos (Santa Cruz; 1:30,000). Fos immunolabeling was apparent in the supraoptic nucleus of the hypothalamus after short-term, but not long-term, Na+ depletion. Fos labeling was not present in the paraventricular hypothalamic nucleus with either protocol, but was present after both protocols in forebrain circumventricular organs (CVOs). Estrogen did not affect fos immunolabeling in either protocol. Thus, independent of estrogen, acute Na+ loss produces transient neural activation in some central areas involved in body Na+ regulation, and sustained activation in CVOs, which monitor circulating hormones and electrolytes. Supported by OCAST HR12-196

Tumor Necrosis Factor‐α Receptor 1 (TNFR1) Knockdown in Subfornical Organ (SFO) Reduces Sympathetic and Hemodynamic Responses to Blood‐Borne TNF‐α

We recently reported that the SFO, a forebrain circumventricular organ that lacks a blood‐brain barrier, plays a major role in mediating sympathetic and hemodynamic responses to blood‐borne proinflammatory cytokines (PICs). Blood‐borne PICs upregulate inflammation and renin‐angiotensin system (RAS) activity in cardiovascular regions of the brain to activate the sympathetic nervous system, but the underlying mechanisms are not fully understood. We tested the contribution of TNFR1 in the SFO to the sympathetic and hemodynamic responses elicited by blood‐borne TNF‐α. Rats received SFO microinjections of a TNFR1 shRNA or scrambled shRNA lentiviral vector, or vehicle. Two weeks later, RT‐PCR showed that TNFR1 shRNA reduced TNFR1 mRNA by 43% (p<0.05) in the SFO but not in the paraventricular nucleus (PVN). Intracarotid artery injection of TNF‐α (200 ng) increased blood pressure, heart rate, and renal sympathetic nerve activity in rats treated with scrambled shRNA or vehicle. These excitatory responses to TNF‐α were attenuated (p<0.05) in rats treated with TNFR1 shRNA. Four hours after TNF‐α injection, mRNA for NF‐κB, cyclooxygenase‐2 and angiotensin II type 1 receptor had increased in both SFO and PVN in rats treated with scrambled shRNA or vehicle. In rats treated with SFO TNFR1 shRNA, the TNF‐α‐induced increases in these parameters were reduced (p<0.05) not only in SFO but also in PVN. These data suggest that blood‐borne TNF‐α acts upon TNFR1 in SFO to upregulate inflammation and RAS activity in SFO, with downstream effects on excitatory mediators in the PVN that contribute to sympathetic excitation. Supported by NIH Grants HL‐073986 and HL‐096671 and Department of Veterans Affairs.

Angiotensin II receptor type‐1a knockdown in subfornical organ prevents sustained increase in mean arterial pressure associated with chronic intermittent hypoxia (686.20)

Sleep apnea (SA) is associated with a sustained increase in mean arterial pressure (MAP) even during waking hours. Chronic intermittent hypoxia (CIH) models the hypoxemia associated with SA and produces elevated MAP during CIH and normoxia. Angiotensin II (Ang II) is implicated in the CIH associated increase in MAP. Subfornical organ (SFO), a forebrain circumventricular organ, lacks blood brain barrier and is a major site for the central effects of circulating Ang II. We investigated the effects of Ang II type 1a receptor knockdown (AT1aRKD) in the SFO on CIH hypertension in adult male rats. Adeno‐associated viral vectors carrying GFP and either AT1aR shRNA or scrambled shRNA (SCM) were injected in SFO. Using laser‐capture microdissection and qRT‐PCR of amino‐allyl RNA, AT1aRKD rats showed decreased SFO AT1aRmRNA in comparison with SCM rats (p<0.05). During light phase, the AT1aRKD rats exposed to CIH(3 min 10% O2 and 3 min room air cycles for 8 h during light phase for 7 d) exhibited significant increases in MAP vs. AT1aRKD‐Normoxia group (p<0.05). During the normoxic dark phase, there was no difference in MAP between CIH and normoxic AT1aRKD rats (p=0.69). SCM‐CIH group showed significant increase in MAP from SCM‐Normoxia group during light phase CIH (p<0.001) and the normoxic dark phase (p<0.001). Our data indicate that AT1aRs in SFO may play a role in the sustained increases in MAP during normoxia associated with CIH.Grant Funding Source: Supported by: P01HL088052‐05

Subfornical Organ Mediates Sympathetic and Hemodynamic Responses to Blood-Borne Proinflammatory Cytokines

Proinflammatory cytokines play an important role in regulating autonomic and cardiovascular function in hypertension and heart failure. Peripherally administered proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), act on the brain to increase blood pressure, heart rate, and sympathetic nerve activity. These molecules are too large to penetrate the blood-brain barrier, and so the mechanisms by which they elicit these responses remain unknown. We tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood-brain barrier, plays a major role in mediating the sympathetic and hemodynamic responses to circulating proinflammatory cytokines. Intracarotid artery injection of TNF-α (200 ng) or IL-1β (200 ng) dramatically increased mean blood pressure, heart rate, and renal sympathetic nerve activity in rats with sham lesions of the SFO (SFO-s). These excitatory responses to intracarotid artery TNF-α and IL-1β were significantly attenuated in SFO-lesioned (SFO-x) rats. Similarly, the increases in mean blood pressure, heart rate, and renal sympathetic nerve activity in response to intravenous injections of TNF-α (500 ng) or IL-1β (500 ng) in SFO-s rats were significantly reduced in the SFO-x rats. Immunofluorescent staining revealed a dense distribution of the p55 TNF-α receptor and the IL-1 receptor accessory protein, a subunit of the IL-1 receptor, in the SFO. These data suggest that SFO is a predominant site in the brain at which circulating proinflammatory cytokines act to elicit cardiovascular and sympathetic responses.

AT1a Receptor Knockdown in the Median Preoptic Nucleus Attenuates the Sustained Component of Hypertension Resulting from Chronic Intermittent Hypoxia

Obstructive sleep apnea produces hypertension and increases sympathetic nerve activity during the hypoxic sleeping events as well as during the waking hours. To model the hypoxemia experienced during sleep apnea, rodents are exposed to chronic intermittent hypoxia (CIH). The median preoptic nucleus (MnPO) is a forebrain region that contributes to this sustained increase in mean arterial pressure (MAP) from CIH. This region integrates information from forebrain circumventricular organs and projects to the paraventricular nucleus to influence sympathetic nerve activity. Our lab has previously shown that in response to CIH there is increased ΔFosB expression in the MnPO and that after a 7 day hypoxia protocol MnPO AT1a receptor mRNA is increased 7 fold whereas AT1b shows no change. In this study, we tested the hypothesis that MnPO AT1a receptors contribute to CIH hypertension using adult male Sprague Dawley rats injected with a neuron specific adenoviral vector with either shRNA to knock down expression of AT1a receptors in the MnPO or a scrambled RNA sequence. All rats showed an increase in MAP during the hypoxic light period, but rats receiving the AT1a knock down did not exhibit the sustained increase in MAP during the normoxic dark phase (P<.05). This data indicates that the AT1a receptor in the MnPO is necessary for the sustained component of hypertension resulting from CIH. P01 HL‐88052

Central Cardiovascular Circuits Contribute to the Neurovascular Dysfunction in Angiotensin II Hypertension

Hypertension, a powerful risk factor for stroke and dementia, has damaging effects on the brain and its vessels. In particular, hypertension alters vital cerebrovascular control mechanisms linking neural activity to cerebral perfusion. In experimental models of slow-developing hypertension, free radical signaling in the subfornical organ (SFO), one of the forebrain circumventricular organs, is critical for the hormonal release and sympathetic activation driving the elevation in arterial pressure. However, the contribution of this central mechanism to the cerebrovascular alterations induced by hypertension remains uncertain. We tested the hypothesis that free radical production in the SFO is involved in the alterations in cerebrovascular regulation produced by hypertension. In a mouse model of gradual hypertension induced by chronic administration of subpressor doses of angiotensin II (AngII), suppression of free radicals in the SFO by overexpression of CuZn-superoxide dismutase (CuZnSOD) prevented the alteration in neurovascular coupling and endothelium-dependent responses in somatosensory cortex induced by hypertension. The SFO mediates the dysfunction via two signaling pathways. One involves SFO-dependent activation of the paraventricular hypothalamic nucleus, elevations in plasma vasopressin, upregulation of endothelin-1 in cerebral resistance arterioles and activation of endothelin type A receptors. The other pathway depends on activation of cerebrovascular AngII type 1 (AT1) receptors by AngII. Both pathways mediate vasomotor dysfunction by inducing vascular oxidative stress. The findings implicate for the first time the SFO and its efferent hypothalamic pathways in the cerebrovascular alterations induced by AngII, and identify vasopressin and endothelin-1 as potential therapeutic targets to counteract the devastating effects of hypertension on the brain.

The role of the subfornical organ in angiotensin II-salt hypertension in the rat.

Hypertension caused by chronic infusion of angiotensin II (Ang II) in experimental animals is dependent, in part, on increased activity of the sympathetic nervous system. This chronic sympathoexcitatory response is amplified by a high-salt diet, suggesting an interaction of circulating Ang II and dietary salt on sympathetic regulatory pathways in the brain. The present study tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ known to be activated by circulating Ang II, is crucial to the pathogenesis of hypertension induced by chronic Ang II administration in rats on a high-salt diet (Ang II-salt model). Rats were randomly selected to undergo either subfornical organ lesion (SFOx) or sham surgery (Sham) and then placed on a high-salt (2% NaCl) diet. One week later, rats were instrumented for radiotelemetric measurement of mean arterial pressure (MAP) and heart rate (HR) and placed in metabolic cages to measure sodium and water balance. Baseline MAP was slightly (but not statistically) lower in SFOx compared with Sham rats during the 5 day control period. During the subsequent 10 days of Ang II administration, MAP was statistically lower in SFOx rats. However, when MAP responses to Ang II were analysed by comparing the change from the 5 day baseline period, only on the fifth day of Ang II was MAP significantly different between groups. There were no differences between groups for water or sodium balance throughout the protocol. We conclude that, although the SFO is required for the complete expression of Ang II-salt hypertension in the rat, other brain sites are also involved.

Angiotensin II, Oxidant Signaling, and Hypertension

It has been 30 years since landmark studies by Johnson and Brody 1 revealed a pivotal role for the forebrain circumventricular organs (CVOs) in experimental hypertension. We have since come to understand that these small midline structures, mounted along the cerebral ventricles and lacking well-formed blood-brain barriers, shoulder a huge responsibility for maintaining the delicate balance of cardiovascular and body fluid homeostases. With their exotic cytology and morphology, including “neuron-like” cells lying free on the ependymal surface and unusually dense and complex fenestrated capillary networks, the CVOs are involved in a remarkable array of homeostatic functions ranging from thirst and salt appetite to vasopressin release and sympathetic outflow.2 With the study by Lob et al in this issue of Hypertension, 3 we must also consider adding to this list the key role that CVOs play in linking central and peripheral mechanisms of hypertension through activation of peripheral T lymphocytes. If proven true, these findings could have broad implications for a unifying hypothesis of how the central nervous system, the vasculature, and possibly other peripheral organs, including the kidney, are involved in the etiology of hypertension. The first of 2 major findings in the study by Lob et al 3 supports and extends previous reports that reactive oxygen species signaling in the subfornical organ (SFO), a key forebrain CVO, is critical in angiotensin II (Ang II)–mediated regulation of blood pressure and hypertension. 4,5 Using an established viral gene transfer approach that induces robust transgene expression nearly exclusively in the SFO, 6 Lob et al3 show that SFO-targeted ablation of endogenous extracellular superoxide dismutase (SOD3), 1 of 3 isozymes in mammals that catalyzes dismutation of superoxide (O2 ), causes a significant elevation in basal blood pressure. In addition, deletion of SOD3 in the SFO increases the sensitivity to systemic Ang II at a dose that does not normally affect blood pressure in mice. These studies using gene deletion lead to a similar general conclusion as was made earlier using gene overexpression, namely, that elevated levels of O2 in the SFO lead to hypertension. What is new and important here is the unmasking of a key role for the extracellular form of SOD. In previous studies, we showed that adenoviral-mediated overexpression of cytoplasmic Cu/Zn SOD or mitochondrial SOD but not SOD3 in the SFO interfered with the pressor effects of Ang II. 4,5 However, as shown earlier and reiterated in the study by Lob et al,3 SOD3 is expressed at high basal levels in SFO. This may explain why additional overexpression of this form of the enzyme failed to inhibit the pressor effects of Ang II in our earlier studies. The use of Cre-loxP technology and selective deletion of endogenous SOD3 in the present study elegantly reveal that extracellular O2 signaling in SFO is also important. Future analysis of the relative expression, distribution, and functional role of these 3 SOD isozymes in SFO will be important in understanding the mechanisms of central

Pathways to hypertension

Hypertension remains a leading risk factor for morbidity and mortality from cardiovascular disease. Elevated sympathetic nerve activity contributes to the development and maintenance of hypertension, but the central mechanisms that lead to chronic increases in sympathetic nerve activity remain unresolved (Guyenet, 2006). Understanding these mechanisms requires detailed anatomical and physiological mapping of the relevant neurocircuitry. Recent studies highlight the importance of the paraventricular nucleus of the hypothalamus (PVN) in mediating acute and chronic increases in sympathetic nerve activity under multiple conditions including hypertension and heart failure (Benarroch, 2005; Guyenet, 2006). However, the PVN seems to have minimal influence on resting sympathetic nerve activity under normal conditions, which raises the important question: what inputs turn on the PVN (Dampney et al. 2005)? Recent investigations are increasing our understanding of the regulation of PVN neuronal activity. In this issue of The Journal of Physiology, Shi et al. (2008) demonstrate that a direct neural connection originating in the organum vasculosum of the laminae terminalis (OVLT) and terminating in the PVN mediates activation of PVN neurons by hyperosmolality. Plasma osmolality is a tightly controlled variable, and hyperosmolality stimulates a number of physiological responses that alter both sodium and water balance in order to return osmolality to within normal limits (Bourque, 2008). Hyperosmolality also stimulates a stress response that includes activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis (Benarroch, 2005; Stocker et al. 2008). The osmoreceptors that mediate these responses are present in multiple locations including the OVLT (Bourque, 2008). The OVLT is a circumventricular organ, meaning that it is exposed to circulating factors including hormones and alterations in plasma osmolality, and also contains neurons that project into the regions of the brain that are protected by the blood–brain barrier. The OVLT mediates, in part, the increases in vasopressin and sympathetic nerve activity that result from elevations in plasma osmolality (McKinley et al. 2001; Shi et al. 2007). Experimentally, hyperosmolality can be induced by high salt intake, and a high salt diet promotes hypertension in susceptible humans and experimental animals (Guyenet, 2006; Osborn, 2007; Stocker et al. 2008). Hyperosmolality can also be induced by dehydration. However, both a high salt diet and dehydration stimulate osmoreceptors throughout the body. The experiments performed by Shi et al. (2008) utilized intracarotid infusion of hypertonic saline to more selectively stimulate forebrain circumventricular organ osmoreceptors including those in the OVLT. They used retrograde tracing techniques to identify neurons that projected directly from the OVLT to the PVN, and they used immunohistochemistry for Fos to identify OVLT neurons that were activated by the hypertonic saline. Combining these three approaches they were able to demonstrate that increases in plasma osmolality directly activate OVLT neurons that project monosynaptically to the PVN. The results of this study are strengthened by two additional aspect of their experimental approach. First, they confirmed that the selected doses of hypertonic saline delivered to the forebrain increased sympathetic nerve activity, which is an effect that is dependent on PVN activation (Chen & Toney, 2001; Antunes et al. 2006). Second, they demonstrated that these doses of hypertonic saline increased arterial pressure in conscious rats, allowing for clear interpretation of the Fos data. The results of this study provide the basis for future studies to determine the physiological role of activating the OVLT to PVN pathway in the development and maintenance of salt-sensitive hypertension. The study also raises some broader questions. (i) What other blood-borne factors can activate the neurons that project directly from the OVLT to the PVN? (ii) Are neuronal pathways that activate the sympathetic nervous system in response to a high salt diet shared with other hypertension risk factors that stimulate sympathetic outflow, such as psychological stress? If so, do they converge in circumventricular organs such as the OVLT, in the PVN or elsewhere? It is likely that multiple converging pathways lead to hypertension, including central neural pathways that drive sustained activation of the sympathetic nervous system. Careful experiments such as those reported by Shi et al. (2008) will help us map these pathways, so we can then determine what turns them on.

Scavenging superoxide selectively in mouse forebrain is associated with improved cardiac function and survival following myocardial infarction

Dysregulation in central nervous system (CNS) signaling that results in chronic sympathetic hyperactivity is now recognized to play a critical role in the pathogenesis of heart failure (HF) following myocardial infarction (MI). We recently demonstrated that adenovirus-mediated gene transfer of cytoplasmic superoxide dismutase (Ad-Cu/ZnSOD) to forebrain circumventricular organs, unique sensory structures that lack a blood-brain barrier and link peripheral blood-borne signals to central nervous system cardiovascular circuits, inhibits both the MI-induced activation of these central signaling pathways and the accompanying sympathoexcitation. Here, we tested the hypothesis that this forebrain-targeted reduction in oxidative stress translates into amelioration of the post-MI decline in myocardial function and increase in mortality. Adult C57BL/6 mice underwent left coronary artery ligation or sham surgery along with forebrain-targeted gene transfer of Ad-Cu/ZnSOD or a control vector. The results demonstrate marked MI-induced increases in superoxide radical formation in one of these forebrain regions, the subfornical organ (SFO). Ad-Cu/ZnSOD targeted to this region abolished the increased superoxide levels and led to significantly improved myocardial function compared with control vector-treated mice. This was accompanied by diminished levels of cardiomyocyte apoptosis in the Ad-Cu/ZnSOD but not the control vector-treated group. These effects of superoxide scavenging with Ad-Cu/ZnSOD in the forebrain paralleled increased post-MI survival rates compared with controls. This suggests that oxidative stress in the SFO plays a critical role in the deterioration of cardiac function following MI and underscores the promise of CNS-targeted antioxidant therapy for the treatment of MI-induced HF.

Right atrial stretch alters fore‐ and hind‐brain expression of c‐fos and inhibits the rapid onset of salt appetite

The inflation of an intravascular balloon positioned at the superior vena cava and right atrial junction (SVC-RAJ) reduces sodium or water intake induced by various experimental procedures (e.g. sodium depletion; hypovolaemia). In the present study we investigated if the stretch induced by a balloon at this site inhibits a rapid onset salt appetite, and if this procedure modifies the pattern of immunohistochemical labelling for Fos protein (Fos-ir) in the brain. Male Sprague-Dawley rats with SVC-RAJ balloons received a combined treatment of furosemide (Furo; 10 mg (kg bw)(-1)) plus a low dose of the angiotensin-converting enzyme inhibitor captopril (Cap; 5 mg (kg bw)(-1)). Balloon inflation greatly decreased the intake of 0.3 m NaCl for as long as the balloon was inflated. Balloon inflation over a 3 h period following Furo-Cap treatment decreased Fos-ir in the organum vasculosum of the lamina terminalis and the subfornical organ and increased Fos-ir in the lateral parabrachial nucleus and caudal ventrolateral medulla. The effect of balloon inflation was specific for sodium intake because it did not affect the drinking of diluted sweetened condensed milk. Balloon inflation and deflation also did not acutely change mean arterial pressure. These results suggest that activity in forebrain circumventricular organs and in hindbrain putative body fluid/cardiovascular regulatory regions is affected by loading low pressure mechanoreceptors at the SVC-RAJ, a manipulation that also attenuates salt appetite.

Distribution of stanniocalcin binding sites in the lamina terminalis of the rat

Stanniocalcin (STC-1), a 50 kDa glycoprotein hormone that regulates calcium/phosphate homeostasis in bony fish and mammals, has been shown to be expressed in central neurons and choroid plexus, and to exert a protective effect against hypercalcemic and hypoxic damage to neurons. Circumventricular organs are known to function in the regulation of ion and body fluid balance. Therefore, the possibility exists that STC-1 may be involved in the regulation of calcium/phosphate and fluid homeostasis through its actions on these central sites. In the present study, the distribution of STC-1 binding sites in forebrain circumventricular organs of the rat were investigated by in situ ligand binding using a stanniocalcin-alkaline phosphatase (STC-AP) fusion protein. Cells exhibiting STC-1 binding sites were found throughout the lamina terminalis. Dense cytoplasmic staining was observed predominantly within ependymal cells lining the anterior third ventricle region (AV3V), as well as cells of the choroid plexus. Additionally, neurons of the organum vasculosum of the lamina terminalis, the dorsal and ventral components of the median preoptic nucleus and the rostral aspects of the subfornical organ exhibited dense STC-1 cytoplasmic staining. STC-1 binding sites were also found in cells of the supraoptic nucleus, suprachiasmatic nucleus and anteroventral preoptic nucleus. These data suggest that STC-1 binding sites localized on the ependymal cells of the AV3V region and neurons of circumventricular organs may be associated with neuronal pathways involved in calcium/phosphate and fluid homeostasis.

Challenged sodium balance and expression of angiotensin type 1A receptor mRNA in the hypothalamus of Wistar and Dahl rat strains

The present study investigates the influence of a chronic high Na + diet (8% Na +) on the expression of the angiotensin type 1A (AT 1A) receptor gene in the lamina terminalis and paraventricular nucleus of the hypothalamus (PVH) in normotensive Wistar (W) rats, as well as in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats. Three weeks of 8% Na + diet led to a higher blood pressure in DS rats compared to DR and W rats. Moreover, the high Na + diet was correlated with a decreased expression of AT 1A receptor mRNA in the median preoptic nucleus (MnPO) and in the PVH of DS rats, compared to DR and W rats. Contrastingly, the AT 1A receptor mRNA expression was not altered by the high Na + diet in the forebrain circumventricular organs of all the rat strains. Interestingly, a furosemide-induced Na + depletion was correlated with an increased expression of AT 1A receptor mRNA in the PVH, MnPO and SFO of both the DS and DR rats. It is concluded that chronic high Na + diet did differently regulate the expression of AT 1A receptor mRNA in two hypothalamic integrative centers for hydromineral and cardiovascular balance (the PVH and MnPO) in DS rats, compared to DR and W rats. However, the AT 1A receptor mRNA expression was similarly regulated in DS and DR rats in response to an acute Na + depletion, suggesting a distinct high Na +-induced regulation of the AT 1A receptor gene in the PVH and MnPO of DS rats.

Both Nox2‐ and Nox4‐containing NAD(P)H oxidases are required for the full vasopressor effects of angiotensin‐II (Ang‐II) in the central nervous system

Recently we showed that Nox2 and Nox4 are abundantly expressed in forebrain circumventricular organs (CVOs), and identified Nox2 as a key player in central Ang-II-induced cardiovascular (CV) responses. However, the contribution of Nox4 to these responses is unknown. Here we used adenovirus-mediated delivery of siRNA targeted to Nox4 (AdsiNox4) to test the hypothesis that this homologue is also important in central Ang-II CV effects. Adult C57 mice underwent CVO gene transfer with AdsiNox4 alone (n=5), AdsiNox 4 + AdsiNox2 (AdsiNox2/4, n=3) or a control siRNA (AdsiGFP, n=3). Telemeters were also implanted for conscious recording of blood pressure (BP) and heart rate (HR). After 1wk recovery, BP and HR effects of ICV injection of Ang-II (200ng, 200nl) were recorded. Baseline BP and HR were not different between groups (BP / HR: AdsiNox4 102±3 / 590±32, AdsiNox2/4 108±5 / 619±58, AdsiGFP 103±2 mmHg / 592±11 bpm, p>.05). Ang-II caused a robust pressor and bradycardic response in AdsiGFP mice (ΔBP / ΔHR: 16±2 / −124±17), but these responses were attenuated in AdsiNox4 mice (ΔBP / ΔHR: 8±1 / −54±24, p<.05). More importantly, combined treatment with AdsiNox2 + AdsiNox4 nearly abolished Ang-II-induced CV responses (ΔBP / ΔHR: 2±1 / −21±15, p<.05). These data establish Nox4 as a key molecule in central Ang-II signaling, and suggest that both Nox4 and Nox2 are necessary for the full CV response profile elicited by Ang-II in the brain.

Differential effects of intravenous hyperosmotic solutes on drinking latency and c-Fos expression in the circumventricular organs and hypothalamus of the rat

Hyperosmotic intravenous infusions of NaCl are more potent for inducing drinking and vasopressin (AVP) secretion than equally osmotic solutions of glucose or urea. The fact that all three solutes increased cerebrospinal fluid osmolality and sodium concentration led the investigators to conclude that critical sodium receptors or osmoreceptors for stimulating drinking and AVP secretion were outside the blood-brain barrier (BBB) in the circumventricular organs (CVOs). We tested an obvious prediction of this hypothesis: that all three solutes should increase c-Fos-like immunoreactivity (Fos-ir) inside the BBB, but that only NaCl should increase Fos-ir in the CVOs. We gave intravenous infusions of 3.0 Osm/l NaCl, glucose, or urea to rats for 11 or 22 min at 0.14 ml/min and perfused the rats for assay of Fos-ir at 90 min. Controls received isotonic NaCl at the same volume. Drinking latency was measured, but water was then removed. Drinking consistently occurred with short latency during hyperosmotic NaCl infusions only. Fos-ir in the forebrain CVOs, the subfornical organ, and organum vasculosum laminae terminalis was consistently elevated only by hyperosmotic NaCl. However, all three hyperosmotic solutes potently stimulated Fos-ir in the supraoptic and paraventricular nuclei of the hypothalamus inside the BBB. Hyperosmotic NaCl greatly elevated Fos-ir in the area postrema, but even glucose and urea caused moderate elevations that may be related to volume expansion rather than osmolality. The data provide strong support for the conclusion that the osmoreceptors controlling drinking are located in the CVOs.