There are a wide variety of commercially available antibodies for labeling microglial cells based on different protein targets, as well as antibodies for the same protein target made in different species. While this array of targets and hosts allows for flexibility in immunohistochemical experiments, it is important to validate that different antibodies provide comparable and accurate immunodetection prior to experimental data collection. We found that a commercially available anti-Iba1 antibody, made in goat, produces irregular staining patterns in specific regions of the mouse brain in both sexes, prompting a further investigation into the phenomenon. This Iba1-goat antibody displayed increased numbers of labeled cells when compared with expression of a CX3CR1-GFP reporter and IHC detection of P2RY12, two common microglial markers. Furthermore, immunodetection by other common anti-Iba1 antibodies made in rabbit and chicken did not display the excessive cell labeling when compared with the CX3CR1-GFP reporter. Upon further investigation, this Iba1-goat antibody was observed to highly colocalize with vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus of the hypothalamus (SON), the two main sites of vasopressin production in the brain. Other anti-Iba1 antibodies made in other species did not show this same colocalization with vasopressin. Finally, this effect was species-specific, as Wistar rats did not display erroneous cell labeling by the Iba1-goat antibody. In sum, the present study employs both qualitative and quantitative data to highlight the importance of validating antibody efficacy and specificity in a region- and species-specific manner.

Vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) are activated by systemic challenges that threaten fluid balance. We previously showed that a systemic salt challenge triggers inverse neurovascular coupling (iNVC) in the SON, in which activity-dependent dendritic AVP release induces parenchymal arteriole (PA) vasoconstriction and local hypoxia. In heart failure (HF), however, the polarity of this salt-evoked response is reversed: microglia-derived adenosine acting on A2A receptors overrides an enhanced AVP-mediated vasoconstriction, producing net vasodilation. Still, whether AVP activation by non-osmotic stimuli engages similar neurovascular mechanisms is unknown. Here, we examined whether hypovolemia induced by intraperitoneal polyethylene glycol (PEG) evokes comparable vascular responses in control and HF rats. In vivo two-photon imaging was used to assess PA diameter in response to PEG in HF rats. Plasma protein and osmolarity were measured using the nanodrop ultra spectrophotometer and osmometer respectively. PEG produced a sustained rise in plasma protein concentration without altering plasma osmolality, confirming induction of isosmotic hypovolemia, and evoked vasoconstriction of SON PA in both control and sham rats. In HF rats, PEG still induced vasoconstriction at 60 min, but this response was attenuated by 90 min despite persistent hypovolemia. These findings indicate that hypovolemia engages a vasoconstrictive neurovascular response consistent with the iNVC previously shown to be AVP-mediated during osmotic stimulation, and that this response remains largely intact in HF. The polarity and temporal dynamics of NVC in HF appear to be stimulus-dependent, shaped both by osmotic-specific recruitment of purinergic pathways and by the altered physiological milieu imposed by HF.

BACKGROUND:The paraventricular nucleus of the hypothalamus (PVN) orchestrates neuroendocrine and autonomic output to maintain systolic blood pressure (SBP). Emerging evidence suggests that the PVN utilizes paracrine signals to modulate neighboring neurons. Here, we test the hypothesis that OXT (oxytocin)-synthesizing neurons of the PVN (PVNOXT) release paracrine signals that regulate SBP via modulation of AVP (arginine vasopressin)-synthesizing neurons of the PVN. METHODS:To test the hypothesis, experiments were conducted ex vivo and in vivo in mice with the expression of ChR2 (channelrhodopsin-2) and EYFP (enhanced yellow fluorescent protein) directed to cells synthesizing OXT. RESULTS:We found >90% of EYFP-neurons were immunolabeled for OXT, and blue light elicited action potentials in these neurons. This confirmed directed/functional expression of ChR2-EYFP within PVNOXT. In vivo optogenetic excitation of PVNOXT increased SBP and elicited bradycardia in OXT-ChR2 (mice expressing EYFP-ChR2 in OXT-containing cells) compared with control OXT-Cre (mice expressing Cre-recombinase directed to the OXT gene) without ChR2. Ganglionic blockade had no effect on the increased SBP, but it abolished the bradycardia. These results suggest that exciting PVNOXT likely recruits a neuroendocrine signal to promote vasoconstriction, thus eliciting the baroreflex to induce bradycardia. Consistent with this interpretation, optogenetic excitation of PVNOXT increased circulating OXT; however, the elevated SBP persisted after administration of the OXT receptor antagonist. Intriguingly, in vitro optogenetic excitation of PVNOXT evoked Ca2+ flux in Chinese hamster ovary cells expressing OXT receptors or vasopressin receptors (V1aR [vasopressin receptor 1a]), suggesting that firing of PVNOXT promotes local release of OXT. Optogenetic excitation of PVNOXT augmented firing of vasopressin-synthesizing neurons of the paraventricular nucleus and tended to increase circulating AVP. Remarkably, systemic or central administration of a V1aR antagonist abolished the increased SBP and bradycardia after excitation of PVNOXT. CONCLUSIONS:Collectively, our results reveal that firing of PVNOXT promotes paracrine release of OXT, which via activation of V1aR(s) expressed on vasopressin-synthesizing neurons of the paraventricular nucleus, drives vasopressin secretion that elevates SBP.

Vasopressin (VP) magnocellular neurosecretory neurons of the hypothalamic supraoptic nucleus (SON) are critical regulators of renal water retention and vascular tone. VP neurons undergo detrimental plastic changes in cardiovascular diseases such as heart failure (HF), resulting in hyperexcitability and thus altered fluid/electrolyte balance. A major intrinsic mechanism that regulates the firing activity of VP neurons is the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium-dependent K+ current (IsAHP). The sAHP is activated by Ca2+ and results in an efflux of K+ from the cell, hyperpolarizing it and throttling firing. Importantly, we previously reported that a blunted sAHP contributes to hyperexcitability of VP neurons in heart failure rats. While the features of the sAHP are well characterized, the identity of the channel underlying the IsAHP remains unknown. Combining patch clamp electrophysiology, pharmacology and immunohistochemistry in Wistar rats, we investigated Intermediate conductance Ca2+-dependent K+ (IK) channels as a potential candidate responsible for carrying the IsAHP. We generated and measured the IsAHP in voltage clamp via 20 Hz trains of 20 square voltage pulses (from -50 to +10) once per minute. After 4 min of baseline recording, we bath applied TRAM-34 (1 μM), a specific IK channel blocker. Blocking IK with TRAM-34 failed to inhibit IsAHP peak amplitude, amplitude at 1 s after stimulus end, or area. Post hoc immunohistochemistry was performed to identify the phenotype of the recorded cell. We observed no inhibitory effect of TRAM-34 on the IsAHP in either VP or OT neurons. We also saw no inhibition of IsAHP (voltage clamp) or sAHP (current clamp) in slices preincubated in TRAM-34 for at least 1 h prior to recording. Conversely, we found that TRAM-34 inhibited isolated whole cell K+ currents, supporting the presence of functional, TRAM-34-sensitive IK channels in SON neurons. Taken together, our results indicate that despite the expression of IK in SON neurons and astrocytes, we observed no evidence of a significant contribution to the sAHP in either OT or VP SON neurons. Future studies will be needed to determine other potential K+ channel candidates contributing to the sAHP in SON neurons.

Until recently, it was widely assumed that oxytocin signaling occurred exclusively through the activation of neuronal oxytocin receptors, with neurons being the primary targets of released oxytocin. However, this view was challenged by the discovery of functional oxytocin receptors in central amygdala astrocytes, which are essential for the proper function of local neuronal microcircuits. Since then, astrocytic oxytocin receptors have been implicated in various aspects of rodent physiology and behavior, yet it remains unclear whether this mechanism is region-specific or widespread across the brain. Here, we provide extensive anatomical data on oxytocin receptor expression in mice and rats, functionally validated through calcium imaging. Based on this mapping and using genetic, calcium imaging and behavioral approaches, we further demonstrate a critical role for oxytocin receptor-expressing astrocytes in the nucleus accumbens in social behavior. In summary, our findings demonstrate that oxytocin receptors are widely expressed in astrocytes across different brain regions. In the nucleus accumbens, these receptors modulate social behavior-an observation with significant implications for the current model of oxytocinergic modulation in the brain.

The nucleus of the solitary tract (NTS) and the area postrema (AP) form a tightly coupled dorsal medullary complex that integrates visceral and humoral signals governing autonomic and cardiometabolic regulation. While their neural interconnections are well characterized, the organization and functional significance of their shared vascular network remain poorly understood. Here, we used in vivo two-photon microscopy in rats, combined with fluorescent vascular labeling and retrograde neuronal tracing, to visualize and quantify blood flow within the AP-NTS microcirculation. We identified direct capillary connections between the two regions, confirming a continuous vascular network previously inferred from ex vivo studies. Analysis of red blood cell trajectories revealed that blood flow across these junctions is predominantly unidirectional, from the NTS toward the AP. Morphometric measurements showed that AP capillaries are nearly twice the diameter of those in the NTS, implying a lower local vascular resistance and providing a structural basis for this directionality. Despite these geometric differences, capillary flow velocities were similar between regions, consistent with active regulation that maintains stable perfusion dynamics. Together, these findings uncover a previously unrecognized, functionally asymmetric vascular pathway within the dorsal medulla. By enabling the directed transfer of diffusible neurohumoral signals from the NTS to the AP, this specialized microvascular network adds a novel layer of communication between two key brainstem autonomic centers and may represent an additional mechanism for the integration of visceral and circulating information.

How neuropeptides act within the neural circuits that control social behavior is not well understood. While the prevailing view is that neuropeptides act through synaptic release and then activation of their canonical receptors on postsynaptic membranes, we investigated the role of a very different form of neuropeptide action in a neural circuit regulating social communication. Specifically, we tested the hypothesis that non‐synaptically released oxytocin (OT) can act via the non‐canonical receptors vasopressin V1a receptors (V1aR) to regulate social communication in Syrian hamsters. Scent marking, a key form of hamster social communication, can be enhanced by the α‐melanocortin stimulating hormone (α‐MSH), which stimulates OT but not arginine‐vasopressin (AVP) release. Here, we employed hypothalamic injections of α‐MSH and the α‐MSH MC4R receptor antagonist MCL‐0020 to determine the role of α‐MSH in the expression of flank marking. To determine if these effects were intracellular calcium (iCa 2+ ) dependent, hamsters were injected with AVP to induce flank marking and with the iCa 2+ antagonist TMB‐8 to test whether it was possible to block this behavioral effect. Further, a highly selective AVP V1a receptor (V1aR) antagonist and an OT receptor (OTR) antagonist were injected into the hypothalamus to investigate the receptor responsible for activating flank marking. Finally, we employed an in vitro hypothalamic slice preparation using “Sniffer cells” biosensors to confirm that α‐MSH induced the release of OT but not AVP. First, we found that the in vivo hypothalamic injection of α‐MSH increased odor‐stimulated scent marking, whereas blockade of its receptor with MCL‐0020 reduced this behavior. Hypothalamic infusion of the iCa 2+ antagonist TMB‐8 significantly reduced both AVP‐induced and α‐MSH‐induced flank marking. Moreover, only the V1aR antagonist, and not the OTR antagonist, significantly decreased scent marking in response to hypothalamic infusion of α‐MSH. Finally, biosensor recordings from hypothalamic slices confirmed that α‐MSH stimulates OT, but not AVP, release. Together, these results demonstrate that α‐MSH triggers non‐synaptic OT release that regulates scent marking via V1aR activation, revealing a novel mechanism by which neuropeptides modulate social behavior.

Vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) are activated by systemic challenges that threaten fluid balance. Beyond their classical activity-dependent release of their neuropeptide cargo into the systemic circulation, these neurons also release AVP somatodendritically, enabling local modulation of neuronal excitability and vascular tone. We previously showed that a systemic salt challenge triggers inverse neurovascular coupling (iNVC) in the SON, in which activity-dependent dendritic AVP release induces parenchymal arteriole vasoconstriction and local hypoxia. In rats with heart failure (HF), however, the polarity of this salt-evoked response is reversed: microglia-derived adenosine acting on A2A receptors overrides an enhanced AVP-mediated vasoconstriction, producing net vasodilation. Still, whether AVP activation by non-osmotic stimuli engages similar neurovascular mechanisms is unknown. Here, we examined whether hypovolemia induced by intraperitoneal polyethylene glycol (PEG) evokes comparable vascular responses in control and HF rats. PEG produced a sustained rise in plasma protein concentration and vasoconstriction of SON parenchymal arterioles in both control and sham rats. In HF rats, PEG still induced vasoconstriction at 60 min, but vascular diameters returned to baseline by 90 min despite persistent hypovolemia. These findings indicate that hypovolemia engages a conserved AVP-mediated iNVC program that remains largely intact in HF, and that the previously described polarity reversal during HF is stimulus-specific, emerging during osmotic but not hypovolemic activation of AVP neurons.

Background:Neurovascular coupling (NVC) is essential for matching cerebral blood flow (CBF) to neuronal activity. While cortical NVC has been studied extensively, particularly in the context of sensory processing, little is known about NVC dynamics in deep brain regions, such as the hypothalamus, especially under disease conditions like heart failure (HF), where impaired cortical NVC has been linked to cognitive decline. Our goal in this study was to investigate salt-induced NVC responses in the hypothalamic supraoptic nucleus (SON) of rats with HF, and to determine the role of microglial purinergic signaling in modulating these responses. Methods:Using in vivo two-photon imaging and real-time oxygen measurements in the SON, we assessed neurovascular responses to a systemic salt challenge in a well-established HF rat model that mimics clinical outcomes observed in the human population. Pharmacological and biosensor approaches were employed to dissect the contribution of key vasoactive mediators. Results:Contrary to our original hypothesis, that HF would exacerbate salt-evoked inverse NVC (iNVC; vasoconstriction and hypoxia) as previously reported by our group in healthy rats, in HF, the NVC response was reversed. Here, salt-induced neuronal activation triggered vasodilation and increased SON pO₂, restoring oxygen levels to those of sham controls. This vasodilation was mediated by adenosine acting on A2A receptors and originated from a putative microglial source. Importantly, a masked, enhanced AVP-mediated vasoconstrictive component was still present, as revealed by biosensor assays, indicating a complex interplay between opposing vasoactive signals during HF. Conclusions:These findings reveal a previously unrecognized microglia-driven purinergic mechanism that overrides AVP-mediated vasoconstriction to restore SON oxygenation during salt challenges in HF. The polarity switch in hypothalamic NVC suggests a region- and disease-specific adaptation with potential relevance to neurohumoral dysregulation in HF.

Relief from psychological stress confers cardio-protection by altering brain activity and lowering blood pressure; however, the neuronal circuits orchestrating these effects are unknown. Here, we used male mice to discern neuronal circuits conferring stress relief and reduced blood pressure. We found that neurons residing in the central nucleus of the amygdala (CeA) expressing angiotensin type 2 receptors (AT2R), deemed CeAAT2R, innervate brain nuclei regulating stress responding. In vivo optogenetic excitation of CeAAT2R lowered blood pressure, and this effect was abrogated by systemic hexamethonium or antagonism of GABA receptors within the CeA. Intriguingly, in vivo optogenetic excitation of CeAAT2R was also potently anxiolytic. Delivery of an AT2R agonist into the CeA recapitulated the hypotensive and anxiolytic effects, but ablating AT2R(s) from the CeA was anxiogenic. The results suggest that the excitation of CeAAT2R couples lowered blood pressure with anxiolysis. The implication is that therapeutics targeting CeAAT2R may provide stress relief and protection against cardiovascular disease.

Interoception broadly refers to awareness of one's internal milieu. Vagal sensory afferents monitor the internal milieu and maintain homeostasis by engaging brain circuits that alter physiology and behavior. While the importance of the body-to-brain communication that underlies interoception is implicit, the vagal afferents and corresponding brain circuits that shape perception of the viscera are largely unknown. Here, we use mice to parse neural circuits subserving interoception of the heart and gut. We determine vagal sensory afferents expressing the oxytocin receptor, hereafter referred to as NDGOxtr, send projections to the aortic arch or stomach and duodenum with molecular and structural features indicative of mechanosensation. Chemogenetic excitation of NDGOxtr significantly decreases food and water consumption, and remarkably, produces a torpor-like phenotype characterized by reductions in cardiac output, body temperature, and energy expenditure. Chemogenetic excitation of NDGOxtr also creates patterns of brain activity associated with augmented hypothalamic-pituitary-adrenal axis activity and behavioral indices of vigilance. Recurrent excitation of NDGOxtr suppresses food intake and lowers body mass, indicating that mechanosensation of the heart and gut can exert enduring effects on energy balance. These findings suggest that the sensation of vascular stretch and gastrointestinal distention may have profound effects on whole body metabolism and mental health.

The supraoptic nucleus (SON) of the hypothalamus plays a key role in the regulation of fluid homeostasis through the synthesis and release of vasopressin (VP). In this population, VP release can occur at both dendritic and axonal sites, allowing for both localized and distal effects such as renal water retention and vasoconstriction. Recent work from our laboratory demonstrated an inverse neurovascular coupling (iNVC) response in which VP cell activation by acute salt loading resulted in dendritic release of VP, leading to localized vasoconstriction and hypoxia. Additionally, we demonstrated that the hypoxic milieu in turn potentiates VP cell activity and increases firing, suggesting that these mechanism constitutes a positive feedback loop that optimized VP activation in order to cope with the homeostatic challenge (Roy et al., Cell Reports 2021). Still, the biophysical mechanism by which the iNVC-hypoxia elicits increased firing in VP neurons has not yet been explored. We hypothesized that hypoxia-induced acidosis, via activation of acid sensitive ion channels (ASICs) (which are present in SON VP neurons; Ohbuchi et al in 2010) is a key mechanism contributing to VP cell excitation in the iNVC positive feedback loop. We investigated this with whole cell patch clamp electrophysiology using eGFP-VP transgenic rats. We recorded from identified SON VP cells and measured deflections in voltage as bath pH was changed from 7.25 to 6.3 (10 mins). Bath solution was either HCL-buffered artificial cerebrospinal fluid (aCSF) aerated with 95% O2/5% CO2 or HEPES-buffered solution (HBS). We observed an excitatory response to the decreases in pH in most SON VP cells recorded (68.75%, n = 11/16). These responses were reversible and, in many cases, repeatable following a second stimulation. To examine cell-type specificity, ongoing studies are focusing on comparing these responses to those obtained from oxytocin cells. Further, we plan to repeat these experiments in the presence of the ASIC-blocker amiloride and determine whether the hypoxia-induced excitatory effect during iNVC is indeed dependent on ASIC-channels. Together, our results support our hypothesis that tissue acidosis (likely via activation of ASIC-channels) contributes to the excitatory feedback loop evoked during salt-induced iNVC in VP neurons. This abstract was supported by the National Heart Lung and Blood Institute of the National Institutes of Health [R01 HL162575-01 (J.E.S.)]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Chronic hypoperfusion is a risk factor for neurodegenerative diseases. However, the sequence of events driving ischemia-induced functional changes in a cell-specific manner is unclear. Methods: To address this gap in knowledge, we used the bilateral common carotid artery stenosis (BCAS) mouse model, and evaluated progressive functional changes to neurons, arterioles, astrocytes, and microglial cells at 14 and 28 days post-BCAS surgery. To assess the neuro-glio-vascular response to an acute ischemic insult, brain slices were superfused with low O2 conditions. Using whole-cell patch-clamp electrophysiology, we measured basic membrane properties (e.g., resting membrane potential, capacitance, input resistance) in cortical pyramidal neurons. The activity of astrocytes was evaluated by monitoring Ca2+ from Aldh1l1-CreERT2; R26-lsl-GCaMP6f mice. Vascular reactivity to low O2 from the BCAS mice was also assessed ex vivo. Results: Our data showed no changes to the basic membrane properties of cortical pyramidal neurons. On the other hand, astrocyte activity was characterized by a progressive increase in the resting Ca2+. Notably, at 14 and 28 days post-BCAS, there was an increased expression of anti-inflammatory-related markers (IL-10, S100A10, TRPA1, and Nrf2). These data suggest that, in young mice, BCAS-induced increases in resting Ca2+ were associated with the expression of neuroprotective signals. Contrary to observations in glial cells, vascular function was impaired post-BCAS surgery, as shown by a blunted vasodilatory response to low O2 and the vasodilatory signal, adenosine. Conclusions: Together, these data suggest that, in young mice, BCAS leads to vascular dysfunction (e.g., impaired vasodilation in parenchymal arterioles), and in the absence of neuronal dysfunction, mild ischemia is associated with the activation of glial-derived neuroprotective signals.

The suprachiasmatic nucleus (SCN) sets the phase of oscillation throughout the brain and body. Anatomical evidence reveals a portal system linking the SCN and the organum vasculosum of the lamina terminalis (OVLT), begging the question of the direction of blood flow and the nature of diffusible signals that flow in this specialized vasculature. Using a combination of anatomical and in vivo two-photon imaging approaches, we unequivocally show that blood flows unidirectionally from the SCN to the OVLT, that blood flow rate displays daily oscillations with a higher rate at night than in the day, and that circulating vasopressin can access portal vessels. These findings highlight a previously unknown central nervous system communication pathway, which, like that of the pituitary portal system, could allow neurosecretions to reach nearby target sites in OVLT, avoiding dilution in the systemic blood. In both of these brain portal pathways, the target sites relay signals broadly to both the brain and the rest of the body.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current ( I NMDA ) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential ( V holding ) −70 mV in low concentration of ECF Mg 2+ ([Mg 2+ ] o ) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX ( I PEAQX ) in both DOCA-H 2 O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current ( I holding ) at V holding −70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg 2+ ] o condition. I ifenprodil was not different in DOCA-H 2 O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

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.

Heart failure (HF) patients suffer from cognitive decline and mood impairments, but the molecular signals and brain circuits underlying these effects remain elusive. The hypothalamic neuropeptide oxytocin (OT) is critically involved in regulating mood, and OTergic signalling in the central amygdala (CeA) is a key mechanism that controls emotional responses including anxiety-like behaviours. Still, whether an altered OTergic signalling contributes to mood disorders in HF remains unknown. To address this, we used an ischaemic rat HF model, along with a highly multidisciplinary approach, to mechanistically study multiple levels of the hypothalamus-to-CeA OTergic circuit in male rats with HF. We aimed to test the hypothesis that sustained activation of the OT system following an infarct leads to depletion of OT content in this pathway, with subsequent changes in OT receptor expression and blunted modulation of local GABAergic circuits. We found that most of OTergic innervation of the CeA originated from the supraoptic nucleus (SON). While no differences in the numbers of SON→CeA OTergic neurons was observed between sham and HF rats, we observed 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 HF rats. We provide the first evidence that male HF rats display perturbations in the hypothalamus-to-amygdala OTergic circuit, laying the foundation for future translational studies targeting either the OT system or GABAergic amygdalar microcircuit to ameliorate mood impairments in rats or patients with chronic HF. KEY POINTS: Heart failure patients suffer from cognitive decline, depression and mood impairments, but the underlying mechanisms remain elusive. Acting within the central amygdala, the neuropeptide oxytocin regulates emotional responses, including anxiety-like behaviours. However, whether changes in oxytocin signalling occurs during heart failure is unknown. In this study, we used an ischaemic rat heart failure model to mechanistically study multiple levels of the hypothalamus-to-amygdala oxytocinergic circuit in this disease. We report an overall blunted oxytocinergic signalling pathway in rats with heart failure, including blunted content and release of oxytocin from axonal terminals, downregulation of neuronal and astrocytic oxytocin receptors, and impaired oxytocin-driven GABAergic synaptic activity within the central amygdala microcircuit of HF rats. These studies shed light on mechanisms that contribute to mood disorders in cardiovascular disease states and help to identify potential molecular targets for their improved treatment.

Calcium (Ca2+) is a critical secondary messenger in all neurons that coordinates important mechanisms of cell physiology. In vasopressin (VP) neurons of supraoptic nucleus (SON) it’s required for somatodendritic release of VP and the slow afterhyperpolarization (sAHP), both of which coordinate neuronal output and thus control stimulus-secretion coupling. Recently, our lab demonstrated that VP sAHPs require endoplasmic reticulum (ER) Ca2+ for activation and that mitochondrial Ca2+ buffering shapes the spatiotemporal trajectory. We also have recent data demonstrating electrotonic segregation between cell compartments. Given the Ca2+ dependence of somatodendritic release and sAHPs, delineation of somatodendritic release from axonal release, and the decoupling of somatic and dendritic electrontonic properties, we hypothesized that calcium dynamics also may be compartmentally segregated as well. Utilizing patch clamp electrophysiology, live calcium imaging, and focal UV uncaging, we probed the spatiotemporal trajectory of rapidly evoked Ca2+ responses in VP neuron somas and dendrites with corresponding membrane potential. UV Ca2+ uncaging at the soma evokes a membrane sAHP and robust increase in cytosolic Ca2+ that propagates into the dendrites. Conversely, Ca2+ uncaged in dendrites propagates bidirectionally from the UV flash but does not penetrate the soma; no membrane hyperpolarization was observed under these conditions. Disabling mitochondrial Ca2+ buffering with Ru360 in the pipette amplifies Ca2+ signasl and sAHPs in somas and dendrites; Ca2+ uncaging in dendrites penetrates soma and can evoke sAHPs under these circumstances. Together, these results suggest that under normal conditions, Ca2+ movement between compartments in VP neurons is unidirectional (soma to dendrite), and that sAHP channels require somatic Ca2+ increases. Moreover, Ca2+ uptake by mitochondria is a critical mechanism that tightly controls the magnitude and progagation of dendritic Ca2+ signals between dendritic and somatic compartments, thus playing an important role in regulating intrinsic mechanisms and ultimately, their neuronal output. NIH K99HL168434 (M.K.K.); R01 HL162575-01 (J.E.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Many neurons including vasopressin (VP) magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus (SON) generate afterhyperpolarizations (AHPs) during spiking to slow firing, a phenomenon known as spike frequency adaptation. The AHP is underlain by Ca2+-activated K+ currents, and while slow component (sAHP) features are well described, its mechanism remains poorly understood. Previous work demonstrated that Ca2+ influx through N-type Ca2+ channels is a primary source of sAHP activation in SON oxytocin neurons, but no obvious channel coupling was described for VP neurons. Given this, we tested the possibility of an intracellular source of sAHP activation, namely, the Ca2+-handling organelles endoplasmic reticulum (ER) and mitochondria in male and female Wistar rats. We demonstrate that ER Ca2+ depletion greatly inhibits sAHPs without a corresponding decrease in Ca2+ signal. Caffeine sensitized AHP activation by Ca2+ In contrast to ER, disabling mitochondria with CCCP or blocking mitochondria Ca2+ uniporters (MCUs) enhanced sAHP amplitude and duration, implicating mitochondria as a vital buffer for sAHP-activating Ca2+ Block of mitochondria Na+-dependent Ca2+ release via triphenylphosphonium (TPP+) failed to affect sAHPs, indicating that mitochondria Ca2+ does not contribute to sAHP activation. Together, our results suggests that ER Ca2+-induced Ca2+ release activates sAHPs and mitochondria shape the spatiotemporal trajectory of the sAHP via Ca2+ buffering in VP neurons. Overall, this implicates organelle Ca2+, and specifically ER-mitochondria-associated membrane contacts, as an important site of Ca2+ microdomain activity that regulates sAHP signaling pathways. Thus, this site plays a major role in influencing VP firing activity and systemic hormonal release.

By transporting products directly from the capillary bed of one region to the capillary bed of another region, vascular portal pathways enable minute amounts of important secretions to reach their specialized targets in high concentrations, without dilution in the systemic circulatory system. For decades there has been only one known portal system in the mammalian brain - that of the pituitary gland, first identified in 1933 (Popa and Fielding, J. Anatomy 1933). This year, we described a second portal pathway in the mouse linking the capillary vessels of the brain's clock suprachiasmatic nucleus (SCN) to those of the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ (Yao et al., Nat. Comm. 2021). A caveat in this initial work was that the direction of blood flow was unknown. To determine whether the SCN signaled the OVLT or vice-versa, we performed in vivo 2-photon imaging in anesthetized eGFP-vasopressin (VP) rats using a recently developed approach (Roy et al., Cell Report 2021) to study blood flow in this portal system. To delineate the SCN microvasculature in vivo, we intravenously infused fluorescent dextrans in anesthetized rats. The SCN-OVLT portal system was identified as Alexa 633 (an artery/arteriole specific dye)-negative vessels originating from a dense SCN capillary network that run rostrally towards the OVLT. These vessels displayed a mean diameter of ~20 μm. Blood flow in the portal vessels was measured by monitoring red blood cell (RBC) movement after intravenous injections with Rho70 kDa. Using kymographs, we found that in all cases, RBCs flowed rostrally, from the SCN towards the OVLT. Importantly, we found than blood flow was significantly higher at night (ZT17-19) compared to daylight (ZT5-7) (p< 0.001), while directionality remained the same (SCN→OVLT). Taken together, our results support the presence of a functional SCN-OVLT portal system in the rat in which blood flows unidirectionally from the SCN towards the OVLT. Moreover, our studies support the notion that blood flow in this system can be regulated. This clock portal system points to entirely new routes and targets for secreted signals from the SCN, restructuring our understanding of its output pathways. Support: NIH HLBI R01HL162575 to JES, AHA916907 to RKR and NSF 1749500 to RS. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.