Raspberry polyphenols target molecular pathways of heart failure.

Approximately 650,000 new cases of heart failure (HF) are diagnosed annually with a 50% five-year mortality rate. HF is characterized by reduced left ventricular (LV) ejection fraction and hypertrophy of the LV wall. The pathophysiological remodeling of the heart is mediated by increased oxidative stress and inflammation. Raspberries are rich in polyphenols which may favorably impact enzymes involved in redox homeostasis while also targeting inflammatory signaling. Thus, the objective of this study was to investigate whether raspberry polyphenols could attenuate HF. Sprague Dawley rats consumed a 10% (w/w) raspberry diet for 7 weeks. At week 3, HF was surgically induced via coronary artery ligation. Hemodynamics and morphology of the heart were assessed. Expression of cardiac proteins involved in oxidative stress, inflammation, apoptosis, and remodeling were examined, and histological analysis was conducted. Additionally, human cardiomyocytes were treated with raspberry polyphenol extract (RBPE) followed by CoCl2 to chemically induce hypoxia. Redox status, apoptosis, and mitochondrial dysfunction were measured. Raspberries attenuated reductions in cardiac function and reduced morphological changes which coincided with reduced toll-like receptor (TLR)4 signaling. Reductions in oxidative stress, apoptosis, and remodeling occurred in vivo. Incubation of cardiomyocytes with RBPE attenuated CoCl2-induced oxidative stress and apoptosis despite pronounced hypoxia-inducible factor (HIF)-1α expression. These data indicate that consumption of raspberries can reduce the underlying molecular drivers of HF; thus, leading to the observed improvements in cardiac functional capacity and morphology. This dietary strategy may be an effective alternative strategy for treating HF. However, further investigation into alternative models of HF is warranted.

Impaired oxytocin signaling in the central amygdala in rats with chronic heart failure.

Aims: Heart failure (HF) patients often suffer from cognitive decline, depression, and mood impairments, but the molecular signals and brain circuits underlying these effects remain elusive. The hypothalamic neuropeptide oxytocin (OT) is critically involved in the regulation of mood, and OTergic signaling in the central amygdala (CeA) is a key mechanism controlling emotional responses including anxiety-like behaviors. Based on this, we used in this study a well-established ischemic rat HF model and aimed to study alterations in the hypothalamus-to-CeA OTergic circuit. Methods and Results: To study potential HF-induced changes in the hypothalamus-to-CeA OTertic circuit, we combined patch-clamp electrophysiology, immunohistochemical analysis, RNAScope assessment of OTR mRNA, brain region-specific stereotaxic injections of viral vectors and retrograde tracing, optogenetic stimulation and OT biosensors in the ischemic HF model. We found that most of OTergic innervation of the central amygdala (CeA) originated from the hypothalamic supraoptic nucleus (SON). While no differences in the numbers of SON→CeA OTertic neurons (or their OT content) was observed between sham and HF rats, we did observe a blunted content and release of OT from axonal terminals within the CeA. Moreover, we report downregulation of neuronal and astrocytic OT receptors, and impaired OTR-driven GABAergic synaptic activity within the CeA microcircuit of rats with HF. Conclusions: Our study provides first evidence that HF rats display various perturbations in the hypothalamus-to-amygdala OTergic circuit, and lays the foundation for future translational studies targeting either the OT system or GABAergic amygdala GABA microcircuit to ameliorate depression or mood impairments in rats or patients with chronic HF.

Angiotensin II-Mediated Neuroinflammation in the Hippocampus Contributes to Neuronal Deficits and Cognitive Impairment in Heart Failure Rats.

BACKGROUND: Heart failure (HF) is a debilitating disease affecting >64 million people worldwide. In addition to impaired cardiovascular performance and associated systemic complications, most patients with HF suffer from depression and substantial cognitive decline. Although neuroinflammation and brain hypoperfusion occur in humans and rodents with HF, the underlying neuronal substrates, mechanisms, and their relative contribution to cognitive deficits in HF remains unknown. METHODS: To address this critical gap in our knowledge, we used a well-established HF rat model that mimics clinical outcomes observed in the human population, along with a multidisciplinary approach combining behavioral, electrophysiological, neuroanatomical, molecular and systemic physiological approaches. RESULTS: Our studies support neuroinflammation, hypoperfusion/hypoxia, and neuronal deficits in the hippocampus of HF rats, which correlated with the progression and severity of the disease. An increased expression of AT1aRs (Ang II [angiotensin II] receptor type 1a) in hippocampal microglia preceded the onset of neuroinflammation. Importantly, blockade of AT1Rs with a clinically used therapeutic drug (Losartan), and delivered in a clinically relevant manner, efficiently reversed neuroinflammatory end points (but not hypoxia ones), resulting in turn in improved cognitive performance in HF rats. Finally, we show than circulating Ang II can leak and access the hippocampal parenchyma in HF rats, constituting a possible source of Ang II initiating the neuroinflammatory signaling cascade in HF. CONCLUSIONS: In this study, we identified a neuronal substrate (hippocampus), a mechanism (Ang II-driven neuroinflammation) and a potential neuroprotective therapeutic target (AT1aRs) for the treatment of cognitive deficits in HF.

Parallel trajectories in the discovery of the SCN-OVLT and pituitary portal pathways: Legacies of Geoffrey Harris.

A map of central nervous system organization based on vascular networks provides a layer of organization distinct from familiar neural networks or connectomes. As a well-established example, the capillary networks of the pituitary portal system enable a route for small amounts of neurochemical signals to reach local targets by traveling along specialized pathways, thereby avoiding dilution in the systemic circulation. The first evidence of such a pathway in the brain came from anatomical studies identifying a portal pathway linking the hypothalamus and the pituitary gland. Almost a century later, we demonstrated a vascular portal pathway that joined the capillary beds of the suprachiasmatic nucleus and a circumventricular organ, the organum vasculosum of the lamina terminalis, in a mouse brain. For each of these portal pathways, the anatomical findings opened many new lines of inquiry, including the determination of the direction of flow of information, the identity of the signal that flowed along this pathway, and the function of the signals that linked the two regions. Here, we review landmark steps to these discoveries and highlight the experiments that reveal the significance of portal pathways and more generally, the implications of morphologically distinct nuclei sharing capillary beds.

Angiotensin-II drives changes in microglia-vascular interactions in rats with heart failure.

Activation of microglia, the resident immune cells of the central nervous system, leading to the subsequent release of pro-inflammatory cytokines, has been linked to cardiac remodeling, autonomic disbalance, and cognitive deficits in heart failure (HF). While previous studies emphasized the role of hippocampal Angiotensin II (AngII) signaling in HF-induced microglial activation, unanswered mechanistic questions persist. Evidence suggests significant interactions between microglia and local microvasculature, potentially affecting blood-brain barrier integrity and cerebral blood flow regulation. Still, whether the microglial-vascular interface is affected in the brain during HF remains unknow. Using a well-established ischemic HF rat model, we demonstrate increased vessel-associated microglia (VAM) in HF rat hippocampi, which showed heightened expression of AngII AT1a receptors. Acute AngII administration to sham rats induced microglia recruitment to the perivascular space, along with increased expression of TNFa. Conversely, administering an AT1aR blocker to HF rats prevented the recruitment of microglia to the perivascular space, normalizing their levels to those in healthy rats. These results highlight the critical importance of a rather understudied phenomenon (i.e., microglia-vascular interactions in the brain) in the context of the pathophysiology of a highly prevalent cardiovascular disease, and unveil novel potential therapeutic avenues aimed at mitigating neuroinflammation in cardiovascular diseases.

Altered PVN-to-CA2 hippocampal oxytocin pathway and reduced number of oxytocin-receptor expressing astrocytes in heart failure rats.

Oxytocinergic actions within the hippocampal CA2 are important for neuromodulation, memory processing and social recognition. However, the source of the OTergic innervation, the cellular targets expressing the OT receptors (OTRs) and whether the PVN-to-CA2 OTergic system is altered during heart failure (HF), a condition recently associated with cognitive and mood decline, remains unknown. Using immunohistochemistry along with retrograde monosynaptic tracing, RNAscope and a novel OTR-Cre rat line, we show that the PVN (but not the supraoptic nucleus) is an important source of OTergic innervation to the CA2. These OTergic fibers were found in many instances in close apposition to OTR expressing cells within the CA2. Interestingly, while only a small proportion of neurons were found to express OTRs (~15%), this expression was much more abundant in CA2 astrocytes (~40%), an even higher proportion that was recently reported for astrocytes in the central amygdala. Using an established ischemic rat heart failure (HF) model, we found that HF resulted in robust changes in the PVN-to-CA2 OTergic system, both at the source and target levels. Within the PVN, we found an increased OT immunoreactivity, along with a diminished OTR expression in PVN neurons. Within the CA2 of HF rats, we observed a blunted OTergic innervation, along with a diminished OTR expression, which appeared to be restricted to CA2 astrocytes. Taken together, our studies highlight astrocytes as key cellular targets mediating OTergic PVN inputs to the CA2 hippocampal region. Moreover, they provide the first evidence for an altered PVN-to-CA2 OTergic system in HF rats, which could potentially contribute to previously reported cognitive and mood impairments in this animal model.

Angiotensin II inhibits the A-type K+ current of hypothalamic paraventricular nucleus neurons in rats with heart failure: role of MAPK-ERK1/2 signaling.

Angiotensin II (ANG II)-mediated sympathohumoral activation constitutes a pathophysiological mechanism in heart failure (HF). Although the hypothalamic paraventricular nucleus (PVN) is a major site mediating ANG II effects in HF, the precise mechanisms by which ANG II influences sympathohumoral outflow from the PVN remain unknown. ANG II activates the ubiquitous intracellular MAPK signaling cascades, and recent studies revealed a key role for ERK1/2 MAPK signaling in ANG II-mediated sympathoexcitation in HF rats. Importantly, ERK1/2 was reported to inhibit the transient outward potassium current (IA) in hippocampal neurons. Given that IA is a critical determinant of the PVN neuronal excitability, and that downregulation of IA in the brain has been reported in cardiovascular disease states, including HF, we investigated here whether ANG II modulates IA in PVN neurons via the MAPK-ERK pathway, and, whether these effects are altered in HF rats. Patch-clamp recordings from identified magnocellular neurosecretory neurons (MNNs) and presympathetic (PS) PVN neurons revealed that ANG II inhibited IA in both PVN neuronal types, both in sham and HF rats. Importantly, ANG II effects were blocked by inhibiting MAPK-ERK signaling as well as by inhibiting epidermal growth factor receptor (EGFR), a gateway to MAPK-ERK signaling. Although no differences in basal IA magnitude were found between sham and HF rats under normal conditions, MAPK-ERK blockade resulted in significantly larger IA in both PVN neuronal types in HF rats. Taken together, our studies show that ANG II-induced ERK1/2 activity inhibits IA, an effect expected to increase the excitability of presympathetic and neuroendocrine PVN neurons, contributing in turn to the neurohumoral overactivity that promotes progression of the HF syndrome.

Inverse neurovascular coupling contributes to positive feedback excitation of vasopressin neurons during a systemic homeostatic challenge.

Neurovascular coupling (NVC), the process that links neuronal activity to cerebral blood flow changes, has been mainly studied in superficial brain areas, namely the neocortex. Whether the conventional, rapid, and spatially restricted NVC response can be generalized to deeper and functionally diverse brain regions remains unknown. Implementing an approach for in vivo two-photon imaging from the ventral surface of the brain, we show that a systemic homeostatic challenge, acute salt loading, progressively increases hypothalamic vasopressin (VP) neuronal firing and evokes a vasoconstriction that reduces local blood flow. Vasoconstrictions are blocked by topical application of a VP receptor antagonist or tetrodotoxin, supporting mediation by activity-dependent, dendritically released VP. Salt-induced inverse NVC results in a local hypoxic microenvironment, which evokes positive feedback excitation of VP neurons. Our results reveal a physiological mechanism by which inverse NVC responses regulate systemic homeostasis, further supporting the notion of brain heterogeneity in NVC responses.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Correction to: Three-dimensional morphometric analysisreveals time-dependent structural changesin microglia and astrocytes in the centralamygdala and hypothalamicparaventricular nucleus of heart failure rats.

An amendment to this paper has been published and can be accessed via the original article.

Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons.

Oxytocin (OT) is a great facilitator of social life but, although its effects on socially relevant brain regions have been extensively studied, OT neuron activity during actual social interactions remains unexplored. Most OT neurons are magnocellular neurons, which simultaneously project to the pituitary and forebrain regions involved in social behaviors. In the present study, we show that a much smaller population of OT neurons, parvocellular neurons that do not project to the pituitary but synapse onto magnocellular neurons, is preferentially activated by somatosensory stimuli. This activation is transmitted to the larger population of magnocellular neurons, which consequently show coordinated increases in their activity during social interactions between virgin female rats. Selectively activating these parvocellular neurons promotes social motivation, whereas inhibiting them reduces social interactions. Thus, parvocellular OT neurons receive particular inputs to control social behavior by coordinating the responses of the much larger population of magnocellular OT neurons.

Three-dimensional morphometric analysis reveals time-dependent structural changes in microglia and astrocytes in the central amygdala and hypothalamic paraventricular nucleus of heart failure rats.

BACKGROUND: Cardiovascular diseases, including heart failure, are the most common cause of death globally. Recent studies support a high degree of comorbidity between heart failure and cognitive and mood disorders resulting in memory loss, depression, and anxiety. While neuroinflammation in the hypothalamic paraventricular nucleus contributes to autonomic and cardiovascular dysregulation in heart failure, mechanisms underlying cognitive and mood disorders in this disease remain elusive. The goal of this study was to quantitatively assess markers of neuroinflammation (glial morphology, cytokines, and A1 astrocyte markers) in the central amygdala, a critical forebrain region involved in emotion and cognition, and to determine its time course and correlation to disease severity during the progression of heart failure. METHODS: We developed and implemented a comprehensive microglial/astrocyte profiler for precise three-dimensional morphometric analysis of individual microglia and astrocytes in specific brain nuclei at different time points during the progression of heart failure. To this end, we used a well-established ischemic heart failure rat model. Morphometric studies were complemented with quantification of various pro-inflammatory cytokines and A1/A2 astrocyte markers via qPCR. RESULTS: We report structural remodeling of central amygdala microglia and astrocytes during heart failure that affected cell volume, surface area, filament length, and glial branches, resulting overall in somatic swelling and deramification, indicative of a change in glial state. These changes occurred in a time-dependent manner, correlated with the severity of heart failure, and were delayed compared to changes in the hypothalamic paraventricular nucleus. Morphometric changes correlated with elevated mRNA levels of pro-inflammatory cytokines and markers of reactive A1-type astrocytes in the paraventricular nucleus and central amygdala during heart failure. CONCLUSION: We provide evidence that in addition to the previously described hypothalamic neuroinflammation implicated in sympathohumoral activation during heart failure, microglia, and astrocytes within the central amygdala also undergo structural remodeling indicative of glial shifts towards pro-inflammatory phenotypes. Thus, our studies suggest that neuroinflammation in the amygdala stands as a novel pathophysiological mechanism and potential therapeutic target that could be associated with emotional and cognitive deficits commonly observed at later stages during the course of heart failure.

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones.

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague-Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual-label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin- or oxytocin-labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post-MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.

Activation of oxytocin neurons in the paraventricular nucleus drives cardiac sympathetic nerve activation following myocardial infarction in rats.

Myocardial infarction (MI) initiates an increase in cardiac sympathetic nerve activity (SNA) that facilitates potentially fatal arrhythmias. The mechanism(s) underpinning sympathetic activation remain unclear. Some neuronal populations within the hypothalamic paraventricular nucleus (PVN) have been implicated in SNA. This study elucidated the role of the PVN in triggering cardiac SNA following MI (left anterior descending coronary artery ligation). By means of c-Fos, oxytocin, and vasopressin immunohistochemistry accompanied by retrograde tracing we showed that MI activates parvocellular oxytocin neurons projecting to the rostral ventral lateral medulla. Central inhibition of oxytocin receptors using atosiban (4.5 µg in 5 µl, i.c.v.), or retosiban (3 mg/kg, i.v.), prevented the MI-induced increase in SNA and reduced the incidence of ventricular arrhythmias and mortality. In conclusion, pre-autonomic oxytocin neurons can drive the increase in cardiac SNA following MI and peripheral administration of an oxytocin receptor blocker could be a plausible therapeutic strategy to improve outcomes for MI patients.