Terry G. Beltz

Neuroendocrine and cytokine profile of chronic mild stress-induced anhedonia

A bidirectional relationship exists between depression and cardiovascular disease. Patients with major depression are more likely to develop cardiac events, and patients with myocardial infarction and heart failure are more likely to develop depression. A feature common to both clinical syndromes is activation of proinflammatory cytokines and stress hormones, including the hypothalamic-pituitary-adrenal axis and the renin-angiotensin-aldosterone system. In the present study we examined the hypothesis that exposure to chronic mild stress (CMS), an experimental model of depression that induces anhedonia in rats, is sufficient to activate the production of proinflammatory cytokines and stress hormones that are detrimental to the heart and vascular system. Four weeks of exposure of male, Sprague-Dawley rats to mild unpredictable environmental stressors resulted in anhedonia which was operationally defined as a reduction in sucrose intake without a concomitant effect on water intake. Humoral assays indicated increased plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), plasma renin activity, aldosterone, and corticosterone in the CMS exposed rats. Tissue TNF-alpha and IL-1beta were increased in the hypothalamus, and TNF-alpha was increased in the pituitary gland. These humoral responses to CMS, associated with anhedonia as an index of depression in the rat, are likely to be associated with neurohumoral mechanisms that may contribute to adverse cardiac events. The findings provide a basis for examining more directly the interactions among the central, endocrine, and immune systems in depression associated with heart disease.

Behavioral and cardiovascular changes in the chronic mild stress model of depression.

Depression is a multifaceted psychological disorder that involves changes in behavior, neuroendocrine function, and physiological responses. The present study investigated multiple behavioral and cardiovascular consequences in the chronic mild stress (CMS) rodent model of depression. Rats were exposed to 4 weeks of CMS followed by 4 weeks of a stress-free recovery period. Sucrose intake, a measure of anhedonia, and spontaneous locomotor activity were measured weekly throughout the study, and cardiovascular function tests were conducted at the completion of the protocol. The results indicate that CMS results in anhedonia and reduced locomotor activity, as well as elevated heart rate (HR), reduced HR variability, and elevated sympathetic cardiac tone. The behavioral effects of CMS recovered to baseline (prestress) levels during the recovery period; however, cardiovascular changes were observed following the recovery of sucrose intake and activity levels. The present findings suggest that behavioral changes that are indicative of anhedonia and locomotor alterations associated with depression are dissociable from long-term cardiovascular changes induced by CMS.

The Brain Renin-Angiotensin System Contributes to the Hypertension in Mice Containing Both the Human Renin and Human Angiotensinogen Transgenes

We have previously shown that mice transgenic for both the human renin and human angiotensinogen genes (RA+) exhibit appropriate tissue- and cell-specific expression of both transgenes, have 4-fold higher plasma angiotensin II (AII) levels, and are chronically hypertensive. However, the relative contribution of circulating and tissue-derived AII in causing hypertension in these animals is not known. We hypothesized that the brain renin-angiotensin system contributes to the elevated blood pressure in this model. To address this hypothesis, mean arterial pressure (MAP) and heart rate were measured in conscious, unrestrained mice after they were instrumented with intracerebroventricular cannulae and carotid arterial and jugular vein catheters. Intracerebroventricular administration of the selective AII type 1 (AT-1) receptor antagonist losartan (10 microgram, 1 microL) caused a significantly greater peak fall in MAP in RA+ mice than in nontransgenic RA- controls (-29+/-4 versus -4+/-2 mm Hg, P<0.01). To explore the mechanism of a central renin-angiotensin system-dependent hypertension in RA+ mice, we determined the relative depressor responses to intravenous administration of the ganglionic blocking agent hexamethonium (5 mg/kg) or an arginine vasopressin (AVP) V1 receptor antagonist (AVPX, 10 microgram/kg). Hexamethonium caused equal lowering of MAP in RA+ mice and controls (-46+/-3 versus -52+/-3, P>0.05), whereas AVPX caused a significantly greater fall in MAP in RA+ compared with RA- mice (-24+/-2 versus -6+/-1, P<0.01). Consistent with this was the observation that circulating AVP was 3-fold higher in RA+ mice than in control mice. These results suggest that increased activation of central AT-1 receptors, perhaps those located at sites involved in AVP release from the posterior pituitary gland, plays a role in the hypertension in RA+ mice. Furthermore, our finding that both human transgenes are expressed in brain regions of RA+ mice known to be involved in cardiovascular regulation raises the possibility that augmented local production of AII and increased activation of AT-1 receptors at these sites is involved.

Novel Effect of Mineralocorticoid Receptor Antagonism to Reduce Proinflammatory Cytokines and Hypothalamic Activation in Rats With Ischemia-Induced Heart Failure

Blocking brain mineralocorticoid receptors (MRs) reduces the high circulating levels of tumor necrosis factor (TNF)-&agr; in heart failure (HF) rats. TNF-&agr; and other proinflammatory cytokines activate neurons in the paraventricular nucleus (PVN) of hypothalamus, including corticotropin-releasing hormone (CRH) neurons, by inducing cyclooxygenase (COX)-2 activity and synthesis of prostaglandin E2 by perivascular cells of the cerebral vasculature. We tested the hypothesis that systemic treatment with a MR antagonist would reduce hypothalamic COX-2 expression and PVN neuronal activation in HF rats. Rats underwent coronary ligation to induce HF, confirmed by echocardiography, or sham surgery, followed by 6 weeks treatment with eplerenone (30 mg/kg per day, orally) or vehicle (drinking water). Eplerenone-treated HF rats had lower plasma TNF-&agr;, interleukin (IL)-1&bgr; and IL-6, less COX-2 staining of small blood vessels penetrating PVN, fewer PVN neurons expressing Fra-like activity (indicating chronic neuronal activation), and fewer PVN neurons staining for TNF-&agr;, IL-1&bgr;, and CRH than vehicle-treated HF rats. COX-2 and CRH protein expression in hypothalamus were 1.7- and 1.9-fold higher, respectively, in HF+vehicle versus sham+vehicle rats; these increases were attenuated (26% and 25%, respectively) in HF+eplerenone rats. Eplerenone-treated HF rats had less prostaglandin E2 in cerebrospinal fluid, lower plasma norepinephrine levels, lower left ventricular end-diastolic pressure, and lower right ventricle/body weight and lung/body weight ratios, but no improvement in left ventricular function. Treatment of HF rats with anticytokine agents, etanercept or pentoxifylline, produced very similar results. This study reveals a previously unrecognized effect of MR antagonism to minimize cytokine-induced central neural excitation in rats with HF.

The Effects of Chronic Fluoxetine Treatment on Chronic Mild Stress-Induced Cardiovascular Changes and Anhedonia

BACKGROUND Depression has a complex bidirectional association with heart disease. Previously we have shown notable cardiovascular changes in the chronic mild stress (CMS) rodent model of depression. Here we investigated the effects of a serotonin-specific reuptake inhibitor on a behavioral index of depression (anhedonia) and cardiac function in rats exposed to CMS. METHODS Male Sprague-Dawley rats were exposed to either 4 weeks of control conditions or CMS, consisting of unpredictable periods of mild stressors, while being treated concurrently with 4 weeks of daily fluoxetine (10 mg/kg, sc) or vehicle. RESULTS Chronic fluoxetine treatment prevented anhedonia in rats exposed to CMS, versus the CMS group treated with vehicle. However, treatment with fluoxetine in the CMS group only partially prevented specific cardiovascular changes associated with CMS, including elevated resting heart rate (HR), exaggerated pressor and HR responses to air jet stress, reduced cardiac output and stroke volume, and HR exaggerated responses to beta-adrenergic receptor blockade. CONCLUSIONS These findings provide evidence that 4 weeks of fluoxetine treatment can prevent behavioral responses and can partially prevent cardiovascular changes associated with CMS, providing insight into the role of serotonin in the link between depression and cardiovascular dysfunction.

The brain Renin-angiotensin system controls divergent efferent mechanisms to regulate fluid and energy balance.

The renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role within individual tissues such as the brain. The brain RAS is thought to control blood pressure through effects on fluid intake, vasopressin release, and sympathetic nerve activity (SNA), and may regulate metabolism through mechanisms which remain undefined. We used a double-transgenic mouse model that exhibits brain-specific RAS activity to examine mechanisms contributing to fluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenal steroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptor blockade. They are also hyperphagic but lean because of a marked increase in body temperature and metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalized metabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluid intake and energy expenditure.

Role of the central nucleus of the amygdala and bed nucleus of the stria terminalis in experimentally-induced salt appetite

The contributions of the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BST) to salt appetite were evaluated with two treatments which induce sodium chloride (NaCl) ingestion. Cumulative 3 h intakes of 2% NaCl after sodium depletion using furosemide, or subcutaneous (s.c.) injections of yohimbine (YOH), were measured in male, Sprague-Dawley rats both before and after electrolytic lesions of the CeA or the BST. Before surgery, sham-lesion and lesion groups drank equivalent amounts of 2% NaCl in response to furosemide depletion and YOH treatment. After surgery, rats with sham lesions increased their intakes of 2% NaCl following YOH while rats with CeA or BST lesions showed significant decreases. Rats with CeA or BST lesions also showed significant decreases in their intake of 2% NaCl after furosemide depletion, while intakes of the sham lesion groups remained unchanged. Lesions of either nucleus virtually eliminated 24 h need-free salt intake. Before and after surgery, all groups drank equivalent amounts of water in response to s.c. angiotensin II and to s.c. hypertonic saline, indicating the lesions specifically affected salt appetite. The results indicate that the CeA and the BST may be important sites for processing inputs mediating salt appetite.

Central interactions of aldosterone and angiotensin II in aldosterone- and angiotensin II-induced hypertension.

Many studies have implicated both angiotensin II (ANG II) and aldosterone (Aldo) in the pathogenesis of hypertension, the progression of renal injury, and cardiac remodeling after myocardial infarction. In several cases, ANG II and Aldo have been shown to have synergistic interactions in the periphery. In the present studies, we tested the hypothesis that ANG II and Aldo interact centrally in Aldo- and ANG II-induced hypertension in male rats. In rats with blood pressure (BP) and heart rate (HR) measured by DSI telemetry, intracerebroventricular (icv) infusions of the mineralocorticoid receptor (MR) antagonists spironolactone and RU28318 or the angiotensin type 1 receptor (AT1R) antagonist irbesartan significantly inhibited Aldo-induced hypertension. In ANG II-induced hypertension, icv infusion of RU28318 significantly reduced the increase in BP. Moreover, icv infusions of the reactive oxygen species (ROS) scavenger tempol or the NADPH oxidase inhibitor apocynin attenuated Aldo-induced hypertension. To confirm these effects of pharmacological antagonists, icv injections of either recombinant adeno-associated virus carrying siRNA silencers of AT1aR (AT1aR-siRNA) or MR (MR-siRNA) significantly attenuated the development of Aldo-induced hypertension. The immunohistochemical and Western blot analyses of AT1aR-siRNA- or MR-siRNA-injected rats showed a marked reduction in the expression of AT1R or MR in the paraventricular nucleus compared with scrambled siRNA rats. When animals from all studies underwent ganglionic blockade with hexamethonium, there was a smaller reduction in the fall of BP in animals receiving icv AT1R or MR antagonists. These results suggest that ANG II and Aldo interact in the brain in a mutually cooperative manner such that the functional integrity of both brain AT1R and MR are necessary for hypertension to be induced by either systemic ANG II or Aldo. The pressor effects produced by systemic ANG II or Aldo involve increased central ROS and sympathetic outflow.

Evidence Supporting a Functional Role for Intracellular Renin in the Brain

The brain renin–angiotensin system is implicated in the regulation of blood pressure (BP) and fluid homeostasis. Recent studies reveal that 2 forms of renin are expressed in the brain of rodents and humans: secreted prorenin and a nonsecreted intracellular form of active renin (icREN). Although the intracellular action of renin has long been postulated, no data supporting its role in BP regulation has been reported. Therefore, we directly evaluated whether this form of renin has physiological implications for BP regulation by characterizing transgenic mice expressing human icREN driven by the glial fibrillary acidic protein (GFAP) promoter and comparing it with similar mice expressing the secreted form of renin. GFAP-icREN mice express hREN primarily in the brain and at the same level of expression as GFAP-secreted prorenin. Unlike the secreted form, which can be detected in cerebrospinal fluid, no human renin could be detected in the cerebrospinal fluid of GFAP-icREN mice. GFAP-icREN mice were then bred with transgenic mice expressing human angiotensinogen, also driven by the GFAP promoter. Double-transgenic mice expressing either the intracellular renin (2.0±0.12 mL/10 g/day) or secreted renin (2.8±0.3 mL/10 g/day) exhibited an increase in drinking volume compared with nontransgenic littermates (1.5±0.1 mL/10 g/day). Both models exhibited an increase in mean arterial pressure (137±5 and 133±8 mm Hg, respectively) compared with control littermates (115±3 mm Hg), which could be rapidly reduced after ICV injection of losartan. These data support the concept of an intracellular form of renin in the brain, which may provoke functional changes in fluid homeostasis and BP regulation.

Salt Appetite: Interaction of Forebrain Angiotensinergic and Hindbrain Serotonergic Mechanisms

Methysergide injected bilaterally into the lateral parabrachial nucleus (LPBN) increases NaCl intake in several models of renin-dependent salt appetite. The present study investigated the role of angiotensin Type 1 (AT1) receptors in the subfornical organ (SFO) on this effect. The intake of 0.3 M NaCl and water was induced by combined administration of the diuretic, furosemide (FURO), and the angiotensin-converting enzyme inhibitor, captopril (CAP). Pretreatment of the SFO with an AT1 receptor antagonist, losartan (1 microgram/200 nl), reduced water intake but not 0.3 M NaCl intake induced by subcutaneous FURO+CAP. Methysergide (4 microgram/200 nl) injected bilaterally into the LPBN increased 0.3 M NaCl intake after FURO+CAP. Losartan injected into the SFO prevented the additional 0. 3 M NaCl intake caused by LPBN methysergide injections. These results indicate that AT1 receptors located in the SFO may have a role in mediating an enhanced sodium intake produced by methysergide treatment.