Oxytocin and Vasopressin Cross Talk Within the Brain Increases Blood Pressure

Abstract

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 (PVN^OXT) 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 PVN^OXT. In vivo optogenetic excitation of PVN^OXT 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 PVN^OXT likely recruits a neuroendocrine signal to promote vasoconstriction, thus eliciting the baroreflex to induce bradycardia. Consistent with this interpretation, optogenetic excitation of PVN^OXT increased circulating OXT; however, the elevated SBP persisted after administration of the OXT receptor antagonist. Intriguingly, in vitro optogenetic excitation of PVN^OXT evoked Ca²⁺ flux in Chinese hamster ovary cells expressing OXT receptors or vasopressin receptors (V1aR [vasopressin receptor 1a]), suggesting that firing of PVN^OXT promotes local release of OXT. Optogenetic excitation of PVN^OXT 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 PVN^OXT.

CONCLUSIONS:

Collectively, our results reveal that firing of PVN^OXT 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.

Novelty and Significance.

What Is Known?

• Neurons synthesizing OXT (oxytocin) in the paraventricular nucleus of the hypothalamus (PVN) influence blood pressure.

• Neurosecretory PVN neurons create paracrine signals by releasing peptides from dendrites.

What New Information Does this Article Contribute?

• Oxytocin-synthesizing neurons of the paraventricular nucleus release OXT in a paracrine manner to affect the activity of neighboring neurons synthesizing AVP (arginine vasopressin) in the PVN.

• This local oxytocinergic signaling activates AVP-synthesizing neurons of the PVN through the V1aR (vasopressin receptor 1a), thereby driving the release of AVP into the systemic circulation.

• Crosstalk between OXT-synthesizing neurons of the PVN and AVP-synthesizing neurons of the PVN increases blood pressure in an AVP-dependent manner in both males and females.

Hypertension, or elevated blood pressure, is a major risk factor for cardiovascular disease, a leading cause of mortality. The PVN orchestrates neuroendocrine and autonomic outputs to maintain blood pressure within a normal physiological range. Emerging evidence from our group indicates that PVN neurons can release their peptidergic cargo locally, which acts in a paracrine manner to coordinate interpopulation communication within the PVN. We found that oxytocin-synthesizing neurons of the paraventricular nucleus drive oxytocinergic paracrine signaling that activates vasopressin-synthesizing neurons of the paraventricular nucleus through the V1aR. This leads to the release of AVP into the systemic circulation, resulting in an increase in blood pressure. This increased blood pressure was observed in both males and females. Our findings may provide novel therapeutic targets for hypertension, particularly preeclampsia, a condition characterized by severe blood pressure elevations during pregnancy.

Meet the First Author, see p e000746

The paraventricular nucleus of the hypothalamus (PVN) is an integrative hub that orchestrates neuroendocrine release and autonomic outflow to maintain cardiovascular function.¹ It is composed of multiple subtypes of neurons, including large magnocellular neurons and small parvocellular neurons that occupy distinct regions of the PVN.² Within the parvocellular region are neuroendocrine neurons that synthesize corticotropin- or thyrotropin-releasing hormones and project to the median eminence to release hormones from the anterior pituitary.^3,4 In addition, parvocellular presympathetic neurons directly project to sympathetic preganglionic neurons, influencing sympathetic outflow to cardiovascular organs.⁵ Magnocellular neurons, on the other hand, comprise two major populations synthesizing either AVP (arginine vasopressin) or OXT (oxytocin), both of which project to the posterior pituitary to secrete their respective peptides into the systemic circulation.⁶

Despite the molecular similarities between these neuropeptides, including their receptors, OXT and AVP neurons have been considered anatomically and functionally distinct.⁷ AVP released into the circulation acts on the kidneys to induce water retention, while also increasing systolic blood pressure (SBP) via direct vasoconstriction.⁸ OXT was first identified as a neuropeptide that plays an essential role during pregnancy, lactation, and myometrial contraction of the uterus in labor.⁹ A growing body of evidence, however, supports a role for parvocellular OXT-synthesizing neurons of the paraventricular nucleus (PVN^OXT) in cardiovascular regulation. For example, PVN^OXT sends excitatory projections to cardiac vagal nerves^10,11 and stimulation of these projections causes reductions in SBP and heart rate (HR) that mimic baroreflex activation,¹² leading to cardioprotective effects in pathologies, such as heart failure.¹³ In addition, neuroanatomical tract tracing revealed that a population of parvocellular PVN^OXT are spinally projecting neurons that activate sympathetic preganglionic neurons,^14,15 thereby elevating SBP and HR.¹⁶ Together, these studies support the role for parvocellular PVN^OXT in SBP regulation via modulation of autonomic outflow. Still, whether magnocellular PVN^OXT influences SBP, and what the underlying mechanisms are, remains to be elucidated.

In addition to releasing OXT and AVP from their axonal terminals, magnocellular PVN neurons can release neuropeptides locally from dendrites in an activity-dependent manner.^17–20 Importantly, work from our group and others demonstrated that this paracrine, wireless communication mediates interneuronal signaling and coordinates the output of distinct PVN axes necessary for the generation of complex homeostatic responses.^17–20 For example, we showed that vasopressin-synthesizing neurons of the paraventricular nucleus (PVN^AVP) stimulate neighboring presympathetic neurons to mount a proper neurohumoral response to a systemic salt challenge.¹⁸ We also revealed a similar wireless-mediated mechanism coordinating neuroendocrine axes with sympathetic outflow to drive secretion of glucocorticoids and elevate SBP.^19,21 Finally, we showed that activation of PVN^OXT stimulates the dendritic release of OXT that results in interneuronal signaling¹⁷ that dampens hypothalamic-pituitary axis activity.^22,23 However, whether neurosecretory PVN^OXT releases paracrine signals to alter SBP remains unknown.

To address this gap and determine whether PVN^OXT release paracrine signals to affect the activity of neighboring neurons to control SBP, we implemented an integrative approach based on the complementary use of (1) genetic reporting, (2) in vivo optogenetics, cardiovascular recordings, and pharmacology, and (3) ex vivo optogenetics, patch-clamp electrophysiology, and Ca²⁺ imaging. We reveal a novel wireless cross talk among PVN^OXT and PVN^AVP that regulates SBP and HR. PVN^OXT stimulation results in the paracrine release of OXT, which stimulates the activity of PVN^AVP via activation of the V1aR (vasopressin receptor 1a). This intra-PVN neurosecretory cross talk increases systemic levels of AVP that elevate SBP. We propose that this novel interneuronal cross talk could be implicated in pathological increases in SBP, particularly during conditions of enhanced PVN^OXT activity, such as pregnancy (ie, preeclampsia).

Methods

Data Availability

The authors declare that all supporting data, research materials, and detailed methods are provided in the Supplemental Material, including the Major Resources Table.

Animal Subjects

All procedures adhere to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at the University of Florida and Georgia State University.

Statistical Analysis

All values are reported as mean±SEM. Statistical analyses were performed in R, and graphs were generated using GraphPad Prism version 9.1.0 (GraphPad Software, San Diego, CA). Data were assessed for normality using both Shapiro-Wilk and Kolmogorov-Smirnov. Data that passed normality tests were analyzed using parametric statistical tests, whereas data that did not pass normality tests or were too small (<10) to assess normality were statistically analyzed using nonparametric statistical tests. Specific statistical testing performed is described in the corresponding figure legends. The null hypothesis was rejected for P<0.05.

Results

Cre-Directed Expression of Channelrhodopsin-2 to the OXT Gene Allows for Selective Optogenetic Stimulation of PVN^OXT

We crossed OXT-Cre mice (mice expressing Cre-recombinase directed to the OXT gene) with mice that express a CAG (CMV [cytomegalovirus] enhancer chicken β-Actin promoter rabbit β-globin)-driven LoxP-flanked stop-codon preceding the light-sensitive excitatory cation channel, ChR2 (channelrhodopsin-2), and EYFP (enhanced yellow fluorescent protein) fusion gene at the Rosa26 locus. This generated OXT-ChR2 mice (mice expressing EYFP-ChR2 in OXT-containing cells), which express the ChR2-EYFP fusion gene in all cells that synthesize OXT (Figure 1A). First, we evaluated the fidelity of the mouse line (Figure 1B). We found that 94% of EYFP-tagged neurons in the PVN colocalized with neurophysin-1, a marker for OXT-synthesizing neurons⁷ (Figure 1C). Next, we validated the functionality of ChR2 with ex vivo whole-cell patch-clamp experiments in brain slices obtained from OXT-ChR2 mice (Figure 1D). We obtained whole-cell patch-clamp recordings from yellow glowing neurons and found that pulses of 488 nm light at 8 Hz evoked action potentials whether given as a single pulse or repetitively (n=39 cells; 13 male, 26 females; 100%; Figure 1D). Next, we investigated whether the evoked firing of PVN^OXT changes SBP and HR in conscious behaving mice. Male OXT-ChR2 mice were implanted with radiotelemetry transmitters and a chronic dwelling fiberoptic targeting the PVN (Figure 1E). After surgical recovery, mice had the fiberoptic connected to a laser-light source and received either 561 nm light (control) or 473 nm light (optogenetic excitation) using a counterbalanced within-subject design. Before illumination, baseline SBP (561 nm; 130.±2.1 mm Hg versus 473 nm: 125.4±11.1 mm Hg) and HR (561 nm: 517±22 bpm versus 473 nm 522±43 bpm) were not significantly different among the conditions. Interestingly, relative to controls, optogenetic excitation of PVN^OXT (10 mW output; 20 ms pulse width; 5 Hz for 10 minutes, 1-minute ON/OFF) significantly increased SBP (Figure 1E). HR tended to increase with optogenetic excitation of PVN^OXT; however, this effect did not reach statistical significance (Figure 1E). Intriguingly, optogenetic excitation of PVN^OXT also produced robust grooming behavior (Video S1). These results suggest that synchronous firing of PVN^OXT at 5 Hz significantly increases SBP. Concurrent to the increased SBP, optogenetic excitation of PVN^OXT also increased grooming behavior, which presents the possibility that the altered cardiovascular responses are secondary to changes in locomotion. To circumvent this potential confound, we conducted follow-up experiments evaluating the cardiovascular responses to optogenetic excitation of PVN^OXT in anaesthetized mice.

Figure 1.

Genetic targeting of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT). A, Schematic depicting the generation of OXT-ChR2 (mice expressing EYFP (enhanced yellow fluorescent protein) and channel-2 rhodopsin (ChR2) in OXT-containing cells) by way of crossing OXT-Cre (mice expressing Cre-recombinase directed to the OXT gene) and Ai32 stop-flox-ChR2-EYFP knock-in mouse lines. B, Confocal scan demonstrating ChR2-EYFP expression throughout the paraventricular nucleus of the hypothalamus (PVN). C, Representative PVN section highlighting colocalization of EYFP and neurophysin-1 (a marker for OXT-synthesizing neurons). Pie chart quantifies colocalization (n=3). D, The in vitro patch-clamp preparation used to evaluate ChR2 functionality in PVN^OXT of OXT-ChR2 mice (mice expressing EYFP-ChR2 in OXT-containing cells). Neurons fire action potentials in response to 488 nm blue light after a single pulse (left) or multiple pulses of varying duration at 8 Hz (middle and right). E, Group data depicting responses of systolic blood pressure (SBP) and heart rate (HR) in conscious behaving male OXT-ChR2 mice following control 561 nm or 473 nm (5 Hz, 20 ms pulse width, 10 mW, 10 minutes, 1-minute ON/OFF) light delivered to the PVN. Box represents period of optogenetic stimulation. ART ANOVA followed by Tukey post hoc test, n=3 per group. CAG indicates CMV (cytomegalovirus) enhancer chicken β-Actin promoter rabbit β-globin; LoxP, locus of X-over P1; ROSA, Reverse Orientation Splice Applicator; and WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element.

Optogenetically Exciting PVN^OXT Increases SBP in Male and Female Mice

To investigate the role that PVN^OXT plays in regulating SBP, we combined in vivo optogenetics with acute cardiovascular recordings. Male and female control (OXT-Cre) and experimental (OXT-ChR2) mice were anaesthetized, and a Millar catheter was implanted into the left common carotid artery (Figure 2A). Mice were then placed in a stereotaxic frame, a microcraniotomy was performed, fiberoptics connected to a laser-light source were inserted above the PVN, and pulses of 473 nm light were delivered to PVN^OXT while cardiovascular parameters were recorded. Before stimulation, baseline SBP and HR were not significantly different between groups (Tables S1 and S2). Relative to OXT-Cre controls, optogenetic excitation of PVN^OXT significantly increased SBP when light was delivered (Figure 2B). This effect occurred in both male and female mice (Figure 2B and 2C). In males, the increased SBP was accompanied by a significant reduction in HR; however, this effect was absent in females (Figure 2B and 2C). Responses to optical stimulation were frequency-dependent in both sexes; however, the increased SBP was more robust in males relative to females (Figure 2D). Moreover, the magnitude of SBP and HR responses in both males and females was not correlated with baseline SBP and HR (Figure S1). We also considered that this sex difference could be attributed to differences in intrinsic membrane properties of the PVN^OXT, namely, a difference in afterhyperpolarizations. However, afterhyperpolarizations evoked by both a 30 Hz current train or an 8 Hz, 120 pulse 488 nm pulse revealed no significant differences in afterhyperpolarization amplitude or area between males and females (Figure S2). Taken together, these results suggest that excitation of PVN^OXT increases SBP in male and female mice.

Figure 2.

Optogenetic excitation of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT) increases systolic blood pressure (SBP) in male and female mice. A, Schematic of the optogenetics-cardiovascular preparation in OXT-Cre (mice expressing Cre-recombinase directed to the OXT gene; control) and OXT–ChR2 (mice expressing EYFP (enhanced yellow fluorescent protein) and channel-2 rhodopsin (ChR2) in OXT-containing cells). B, Representative raw traces of pulsatile blood pressure (BP) and heart rate (HR) in male OXT-Cre and OXT-ChR2 mice after optical stimulation (473 nm, 30 Hz, 20 ms pulse width, 10 mW, 1 minute). C, Group data depicting time course changes of SBP and HR upon optical stimulation in male and female OXT-Cre and OXT-ChR2 mice. D, Group data (left) and linear regression analysis (right) illustrating frequency-dependent responses of SBP and HR in male and female OXT-ChR2 mice. Error bars indicate SEM. Blue box represents period of optogenetic stimulation. ART ANOVA followed by Tukey post hoc test (C), Kruskal-Wallis test followed by Dunn multiple comparison test (D). n=4 (male OXT-Cre), 9 (male OXT-ChR2), 5 (female OXT-Cre), and 7 (female OXT-ChR2).

Optogenetic Inhibition of PVN^OXT Decreases SBP

Next, we used optogenetic inhibition to test the hypothesis that PVN^OXT are necessary for the maintenance of basal SBP. We bilaterally delivered Cre-inducible adeno-associated virus synthesizing EYFP or the light-gated inhibitory chloride-channel SwiChRca (step-waveform inhibitory channel channelrhodopsin) into the PVN of OXT-Cre mice (Figure 3A). Two to three weeks later, coronal slices through the PVN were obtained for ex vivo whole-cell patch-clamp recordings from PVN^OXT. We successfully opened the SwiChRca channel with pulses of 488 nm (Figure 3B). SwiChRca neurons sustained inhibition during 488 nm stimulation. These results show the feasibility of using SwiChRca to optogenetically inhibit PVN^OXT.

Figure 3.

Optogenetic inhibition of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT) decreases systolic blood pressure (SBP) in male mice. A, Schematic of the optogenetics-cardiovascular preparation in male OXT-Cre (mice expressing Cre-recombinase directed to the OXT gene) mice that received Cre-inducible adeno-associated viruses (AAVs) to direct the expression of EYFP (enhanced yellow fluorescent protein; OXT-EYFP; control) and SwiChRca (OXT-SwiChRca; experimental) to PVN^OXT. B, left, EYFP-SwiChRca-positive neurons visualized in an ex vivo acute slice. Right, Gap-free current clamp recording from an EYFP-SwiChRca-positive neuron. 488 nm light at 0.06 Hz sustained inhibition of action potential firing, significantly hyperpolarizing neurons, linear mixed effects model, n=12 cells in 3 mice. C, Representative raw traces of pulsatile blood pressure (BP) and heart rate (HR) in OXT-EYFP and OXT-SwiChRca mice after the illumination of 473 nm (0.06 Hz, 500 ms pulse, 10 mW, 5 minutes). D, Group data depicting the time course of SBP and HR upon optical inhibition in male OXT-EYFP and OXT-SwiChRca mice. Error bars indicate SEM. Blue box represents the period of optogenetic inhibition. ART ANOVA followed by Tukey post hoc test, n=7 (EYFP), 9 (SwiChRca). ITR indicates inverted terminal repeat; SwiChRca indicates step-waveform inhibitory channel channelrhodopsin; and WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element.

In a separate group of OXT-Cre male mice, we again bilaterally delivered Cre-inducible adeno-associated viruses synthesizing EYFP (control mice) or EYFP and SwiChRca (experimental mice) into the PVN. In vivo optogenetics was done as in the previous experiment (Figure 3A). Baseline SBP and HR before stimulation were not significantly different between groups (Table S3). Compared with controls expressing only EYFP, optogenetic inhibition of PVN^OXT significantly reduced SBP (Figure 3C and 3D). No significant differences in HR were observed between groups. Taken together, these results suggest that PVN^OXT are required for the maintenance of basal SBP.

Excitation of PVN^OXT Does Not Affect SBP via the Autonomic Nervous System

The optogenetic experiments revealed that PVN^OXT tonically control SBP; thus, we performed follow-up experiments to interrogate underlying mechanisms. Because the PVN contains presympathetic neurons,¹⁹ our experiments tested the hypothesis that PVN^OXT control SBP through the autonomic nervous system. Male OXT-ChR2 mice underwent anesthetized optogenetic excitation and cardiovascular recordings as described (Figure 4A). Pulses of 473 nm light were delivered to PVN^OXT before and after the peripheral administration of the nicotinic receptor antagonist, hexamethonium (30 mg/kg).²⁴ Baseline SBP and HR were reduced after hexamethonium as expected with the blockade of autonomic transmission (Table S4). The SBP and HR responses to hexamethonium are shown in Figure S3. Exciting PVN^OXT before hexamethonium significantly increased SBP; however, contrary to our hypothesis, this effect persisted in the presence of hexamethonium (Figure 4B and 4C). As observed previously, a decrease in HR accompanied the optogenetically induced elevation in SBP; however, this was completely abolished by hexamethonium (Figure 4B and 4C). Importantly, the optogenetic stimulation of PVN^OXT after the control vehicle (eg, 0.9% saline) elicited a robust pressor response followed by bradycardia (Figure S4). These results suggest that autonomic mechanisms do not contribute to the increased SBP that follows optogenetic excitation of PVN^OXT, but intact autonomic outflow is required for the reduced HR.

Figure 4.

Pressor responses to optogenetically activating OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT) are not sympathetically mediated. A, Schematic of pharmacological manipulation in the optogenetics-cardiovascular preparation in male OXT–ChR2 (mice expressing EYFP (enhanced yellow fluorescent protein) and channel-2 rhodopsin (ChR2) in OXT-containing cells). B, Representative raw traces of pulsatile blood pressure (BP) and heart rate (HR) in male OXT-ChR2 mice during optical stimulation (473 nm, 30 Hz, 20 ms pulse width, 10 mW, 1 minute) before (left) and after (right) IP administration of hexamethonium (30 mg/kg). C, Group data of systolic blood pressure (SBP; left) and HR (right) responses during 473 nm light illumination before and after hexamethonium. Error bars indicate SEM. Blue box represents period of optogenetic stimulation. ART ANOVA followed by Tukey post hoc test, n=4 per group.

Optogenetic Stimulation of PVN^OXT Elicits Elevations in SBP That Are Not Dependent on OXT Receptors

Some PVN^OXT are magnocellular neurons that project to the posterior pituitary and release OXT into the systemic circulation. Therefore, it is possible that optogenetic excitation of PVN^OXT promotes the systemic release of OXT which acts on OTRs (OXT receptors) to increase SBP. We, therefore, performed follow-up experiments to test the hypothesis that PVN^OXT regulates SBP through a neuroendocrine mechanism that is mediated by OTRs. First, we performed anatomic experiments to reveal the pattern of neuronal activation associated with optogenetic excitation of PVN^OXT. Pulses of 473 nm light were delivered to PVN^OXT in conscious behaving male OXT-Cre and OXT-ChR2 mice. Ninety-minutes later, mice were anesthetized, transcardially perfused, and brains were collected and processed for Fos immunohistochemistry, a marker of neuronal activation.²⁵ Relative to OXT-Cre control mice, delivery of 473 nm light to OXT-ChR2 mice significantly increased the number of PVN^OXT labeled for Fos (Figure 5A and 5B). Subsequent experiments evaluated whether this increased neuronal activation was predictive of elevated circulating levels of OXT. Male OXT-Cre and OXT-ChR2 were anesthetized and subsequently implanted with fiberoptics targeting the PVN and pulsed with 473 nm light. Three-minutes later, plasma samples were collected. Relative to controls, optogenetic excitation of PVN^OXT in OXT-ChR2 mice significantly increased plasma levels of OXT (Figure 5C). We conducted additional experiments to test the hypothesis that the increased SBP that accompanies optogenetic excitation of PVN^OXT results from elevated circulating OXT acting on OTRs. Toward this end, we combined in vivo optogenetics with pharmacology as previously described (Figure 5D). Pulses of 473 nm light were delivered to PVN^OXT before or after the peripheral administration of an OTR antagonist (OTA; L-371 257; 1 mg/kg). Baseline SBP and HR were not significantly different before or after the administration of L-371 257 (Table S5). The SBP and HR response to L-371 257 are shown in Figure S3. Optogenetic excitation of PVN^OXT before the administration of the OTA increased SBP and reduced HR as observed in the previous experiments (Figure 5E and 5F). Again, contrary to our hypothesis, OTR blockade had no effect on the SBP or the HR responses to optogenetic excitation of PVN^OXT (Figure 5E and 5F). Collectively, these results suggest that activation of PVN^OXT elevates circulating levels of OXT; however, it is unlikely that elevated OXT acts on OTRs to increase SBP during optogenetic excitation of PVN^OXT.

Figure 5.

Pressor responses to optogenetically activating OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT) are not mediated by OTRs (OXT receptors). A, Representative images depicting colocalization of Fos with neurophysin-1-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) of male OXT-Cre mice (mice expressing Cre-recombinase directed to the OXT gene; left; control) and OXT-ChR2 (mice expressing EYFP-ChR2 in OXT-containing cells; right; experimental) after 473 nm light illumination (30 Hz, 20 ms pulse width, 10 mW, 1 minute). Yellow arrows indicate colocalized cells. B, Quantification of Fos and neurophysin-1 colocalization. P=0.0286; Mann-Whitney U test. n=4 per group. C, Plasma OXT levels of male OXT-Cre and OXT-ChR2 mice following optogenetic stimulation. P=0.0001; Mann-Whitney U test. n=9 (OXT-Cre), 14 (OXT-ChR2). D, Schematic of pharmacological manipulation in the optogenetics-cardiovascular preparation in male OXT-ChR2. E, Representative raw traces of pulsatile blood pressure (BP) and heart rate (HR) in male OXT-ChR2 mice during optogenetic excitation before (left) or after (right) IP administration of L-371 2571 (OTR antagonist; 1 mg/kg). F, Group data of systolic blood pressure (SBP; left) and HR (right) in male OXT-ChR2 undergoing optogenetic stimulation before or after OTR blockade (IP). Blue box represents period of optogenetic stimulation. ART ANOVA followed by Tukey post hoc test, n=9 per group.

Optogenetic Activation of PVN^OXT Leads to the Local Release of OXT That Binds to OTRs or V1aRs

To date, our experiments demonstrated that PVN^OXT increases SBP through actions that are independent of the autonomic nervous system or OTR stimulation. Therefore, we conducted experiments to determine whether excitation of PVN^OXT recruits paracrine hormone signaling that acts within the PVN²⁶ to increase SBP. Initial experiments utilized sniffer cells plated onto our ex vivo slice preparation. Sniffer cells are Chinese hamster ovarian cells overexpressing either OTRs (CHO sniffer cells containing human Oxytocin receptors [Sniffer_OTR]) or V1aRs (CHO sniffer cells containing human V1aRs [Sniffer_V1aR]), transfected with the calcium sensor R-GECO (Red shifted-Genetically Encoded Calcium Indicator) to detect endogenous peptide release.²⁷ Thus, sniffer cells may be used to detect endogenous peptide release in the brain slice. Male OXT-ChR2 mice were euthanized, slices through the PVN were obtained, and Sniffer_OTR cells were plated over PVN^OXT (Figure 6A). Optogenetic stimulation of PVN^OXT generated calcium responses in Sniffer_OTR cells (Figure 6B and 6C). Furthermore, we observed consistent detection of endogenously released OXT in Sniffer_OTR cells plated on the PVN (45.3%; Figure 6D and 6E). As a control, Sniffer_OTR cells were plated on the cortex (layers II–V), and no detectable calcium responses after blue light stimulation were observed (Figure 6D). Interestingly, when Sniffer_V1aR cells were plated on the PVN, we detected calcium responses to PVN^OXT stimulation, albeit at much less efficient rates than Sniffer_OTR, as demonstrated by the 5.5% response rates (Figure 6D). These results indicate that PVN^OXT activation results in detectable and local release of endogenous OXT that is able to bind to both OTRs and V1aRs.

Figure 6.

Optogenetic excitation of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT) evokes the release of OXT that binds to V1aR (vasopressin receptor 1a). A, Representative confocal images of EYFP (enhanced yellow fluorescent protein)–ChR2 (channelrhodopsin-2)–positive PVN^OXT (left), CHO sniffer cells containing human oxytocin receptors (Sniffer_OTR) R-GECO signal (middle), and a merged image (right). B, Pseudo-color images of Sniffer_OTR before (left) and after (right) 488 nm light illumination. Arrowheads mark cells that responded to optogenetic excitation of PVN^OXT. C, Representative F/F₀ calcium traces corresponding to the arrowheads in B, demonstrating Sniffer_OTR responses to PVN^OXT activation. D, Pie charts summarizing response rates of Sniffer_OTR (left; n=6) and Sniffer_V1aR (CHO sniffer cells containing human V1aRs; middle; n=3 animals, 127 cells) plated in paraventricular nucleus of the hypothalamus (PVN), and Sniffer_OTR plated in cortex (right; n=4 animals, 61 cells), after 488 nm light illumination. E, Quantitative analysis of Sniffer_OTR responses in the PVN following blue light stimulation. OTR indicates oxytocin receptors; and R-GECO, Red shifted-Genetically Encoded Calcium Indicator.

Optogenetic Stimulation of PVN^OXT Excites Putative PVN^AVP

Prior studies established that AVP-synthesizing neurons of the PVN (PVN^AVP) express V1aR(s).²⁸ This, in conjunction with our results revealing that optogenetic excitation of PVN^OXT promotes local release of OXT that binds to V1aR(s), presents the possibility that PVN^OXT activates PVN^AVP by releasing OXT that stimulates V1aR(s). To address this potential for cross talk among PVN^OXT and PVN^AVP, we performed ex vivo whole-cell patch-clamp experiments. Briefly, male OXT-ChR2 were euthanized to obtain coronal sections through the PVN (Figure 7A). We patched nonglowing EYFP-negative neurons and distinguished magnocellular from parvocellular neurons through their electrophysiological properties (Figure 7B).^29,30 Thus, we patched on EYFP-negative magnocellular neurons (putative PVN^AVP) that were in proximity to glowing EYFP-positive neurons (PVN^OXT; Figure 7C). After that, we stimulated PVN^OXT by illuminating with 30, 60, and 120 pulses of blue light (Figure 7D). We observed that 30 pulses produced a small, inconsistent upregulation in firing frequency (0.05±0.02 Hz) compared with 60 (0.07±0.03) and 120 pulses (0.36±0.17) in putative PVN^AVP (Figure 7E). Furthermore, the 120-pulse stimulation increased the firing of 31 of 37 putative PVN^AVP with a response latency of 208.5±21 s (Figure 7E). This suggests that PVN^OXT requires high pulses and sustained stimulation to initiate a cross talk that increases the firing of putative PVN^AVP. Taken together, our results indicate a potential neuroendocrine cross talk between PVN^OXT and PVN^AVP may mediate the SBP responses to the optogenetic activation of PVN^OXT.

Figure 7.

Stimulation of oxytocin-synthesizing neurons of the paraventricular nucleus (PVN^OXT) evokes cross talk with AVP (arginine vasopressin) neurons. A, Schematic of the ex vivo whole-cell patch-clamp preparation in male OXT–ChR2 (mice expressing EYFP (enhanced yellow fluorescent protein) and channel-2 rhodopsin (ChR2) in OXT-containing cells). B, EYFP (enhanced yellow fluorescent protein)-negative neurons in the paraventricular nucleus of the hypothalamus (PVN) were characterized as magnocellular vs parvocellular neurons based on the presence of low-threshold hyperpolarization-induced depolarization (arrow) in parvocellular neurons. C, Representative image of nonglowing EYFP-negative magnocellular neurons (putative AVP neuron) surrounded by EYFP-ChR2 positive PVN^OXT. D, Representative traces from putative AVP magnocellular neurons patched in proximity to EYFP-ChR2 positive neurons, in response to ChR2 stimulation by 488 nm light illumination at 8 Hz for 30×, 60×, and 120× pulses. E, Changes in firing (ΔHz) observed after ChR2 stimulation for 30×, 60×, and 120× pulses (top left; F_{[2, 12.90]}=6.27, P=0.0125; linear mixed effects model, n=6 cells, 3 animals followed by Tukey pairwise comparison. 30 stim vs 120 stim t₍₁₅₎=3.406; P=0.0183). Firing frequency changes at baseline and after 120× pulses of ChR2 stimulation (top right; t_[36.00]=2.78, P=0.00866; linear mixed effects model, n=37 cells, 18 animals). Distribution of change in firing frequency (ΔHz) after ChR2 stimulation (bottom left). Latency from ChR2 onset to changes in firing frequency (bottom right).

Excitation of PVN^OXT Increases SBP Through Neuroendocrine Cross Talk Mediated by V1aR(s)

Follow-up experiments tested the hypothesis that neuroendocrine cross talk between PVN^OXT and PVN^AVP mediates the increased SBP that results from optogenetic activation of PVN^OXT. First, we investigated whether optogenetic activation of PVN^OXT elicits consequent activation of PVN^AVP and the systemic release of AVP. OXT-Cre and OXT-ChR2 male mice were used for either dual neurophysin 2 (marker for AVP-synthesizing neurons⁷) and Fos immunohistochemistry or measurement of plasma levels of AVP after optogenetic excitation of PVN^OXT. Optogenetic excitation of PVN^OXT significantly increased Fos immunohistochemistry within neurophysin 2-expressing neurons of the PVN when compared with that of controls (Figure S5A and S5B). Plasma levels of circulating AVP also tended to be higher in OXT-ChR2 mice relative to controls (Figure S5C). Finally, to rule out the possibility that inadvertent expression of ChR2 in PVN^AVP underlies these effects, we evaluated whether neurons expressing the EYFP marker for ChR2 also synthesize the neurophysin 2 marker for AVP. We found the vast majority (>90%) of EYFP-expressing neurons in the PVN did not express neurophysin 2 (Figure S5D and S5E). These collective results suggest that optogenetic excitation of PVN^OXT promotes activation of PVN^AVP and the systemic release of AVP.

So far, our experiments have demonstrated that PVN^OXT evokes elevations in SBP independent of the autonomic nervous system that are likely mediated through neuroendocrine release that promotes vasoconstriction to elicit the baroreflex. Although the optogenetic stimulation of PVN^OXT elevates circulating levels of OXT, SBP responses were not mediated through OTRs. Given that PVN^OXT stimulation activates PVN^AVP, leading to the systemic release of AVP, and that AVP induces vasoconstriction and consequent baroreflex activation,³¹ we combined in vivo optogenetics with pharmacology to investigate whether the cross talk between PVN^OXT and PVN^AVP mediates the SBP responses through V1aR(s). Male OXT-ChR2 mice were anesthetized for our acute in vivo optogenetic-cardiovascular preparation as described (Figure 8A). This time, pulses of 473 nm light were delivered to PVN^OXT before and after the peripheral administration of a V1aR antagonist (SR-49059; 2 mg/kg). Baseline SBP and HR were not significantly different before or after the administration of SR-49059 (Table S6). Responses in SBP and HR after the administration of SR-49059 are reported in Figure. S3. Similar to the previous experiments, optogenetic excitation of PVN^OXT before the administration of SR-49059 increased SBP and reduced HR (Figure 8A). In contrast to the null effects of the OTR blockade, V1aR antagonism completely abolished these responses (Figure 8A). These results support the notion that optogenetic excitation of PVN^OXT elevates circulating levels of AVP that act on peripheral V1aR to increase SBP.

Figure 8.

Optogenetic excitation of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVN^OXT) evokes cross talk with arginine vasopressin-synthesizing neurons of the paraventricular nucleus (PVN^AVP) to elicit systolic blood pressure (SBP) elevations mediated by V1aR (vasopressin receptor 1a). A, left, Schematic of systemic SR-49059 (V1aR antagonist) administration in the optogenetics-cardiovascular preparation in male OXT–ChR2 (mice expressing EYFP (enhanced yellow fluorescent protein) and channel-2 rhodopsin (ChR2) in OXT-containing cells). Right (top), Representative blood pressure (BP) and heart rate (HR) traces in male OXT-ChR2 mice during 473 nm stimulation (30 Hz, 20 ms pulse width, 10 mW, 1 minute) before and after IP. SR-49059 (2 mg/kg). Right (bottom), Group SBP and HR data. ART ANOVA followed by Tukey post hoc test, n=5 per group. B, left, A schematic of the paraventricular nucleus of the hypothalamus (PVN) microinjection of SR-49059 during the optogenetics-cardiovascular preparation in male OXT-ChR2 mice. Right, Group data of SBP and HR in male OXT-ChR2 mice during 473 nm stimulation after vehicle (dimethyl sulfoxide [DMSO]) or V1aR blockade. ART ANOVA followed by Tukey post hoc test, n=5 per group. C, Schematic of the ex vivo whole-cell patch-clamp preparation in male OXT-ChR2 mice. D, Group data demonstrating that OTRs (OXT receptors) blockade (L-371 257) did not alter firing frequency, whereas V1aR blockade (SR-49059) abolished firing in patched putative PVN^AVP following 488 nm illumination. P values are linear mixed effects model pairwise comparisons, n=22 cells from 13 mice. E, Averaged firing frequency over time of neurons in the presence of either SR-49059 or L-371 257.

Next, we combined in vivo optogenetics with pharmacology to investigate whether the cross talk between OXT and AVP neurons within the PVN mediates the SBP responses through V1aR(s). Male OXT-ChR2 mice were anaesthetized for our acute in vivo optogenetic-cardiovascular preparation as described. This time, pulses of 473 nm light were delivered to PVN^OXT after the microinjection of a V1aR antagonist (SR-49059; 2 mmol/L) or a vehicle control (2% dimethyl sulfoxide) into the PVN using a dual-fiberoptic injector as previously described.¹⁹ Similar to the previous experiments, optogenetic excitation of PVN^OXT after the PVN microinjection of the vehicle control increased SBP and reduced HR (Figure 8B). Fascinatingly, V1aR antagonism within the PVN completely abolished these responses (Figure 8B).

To provide further evidence for this cross talk within the PVN, we performed ex vivo electrophysiological recordings in male OXT-ChR2 mice (Figure 8C). After obtaining brain slices through the PVN, we recorded from EYFP-negative magnocellular neurons (putative PVN^AVP) and illuminated blue light before or after the bath application of an OTA (L-371 257; 0.1 µmol/L) or a V1aR antagonist (SR-49059; 0.1 µmol/L; Figure 8D). We found that optogenetic stimulation of PVN^OXT with 488 nm at 8 Hz for 120× pulses increased firing of putative PVN^AVP in the presence of OTA; however, there was no statistically significant difference observed in firing during V1aR blockade (Figure 8D and 8E). Taken together, our studies reveal that excitation of PVN^OXT promotes the release of OXT that binds to V1aR(s) expressed on PVN^AVP. This neuroendocrine cross talk elicits the firing of PVN^AVP and the systemic release of AVP, which increases SBP by activating peripheral V1aR(s) (Graphic Abstract).

Discussion

In the current study, we investigated whether intranuclear PVN^OXT paracrine signals regulate SBP. We report that in vivo optogenetic excitation of PVN^OXT increased SBP in male and female mice. Interestingly, ganglionic blockade had no effect on the increased SBP elicited by optogenetic activation of PVN^OXT. This ruled out an autonomic-mediated change in SBP, suggesting that exciting PVN^OXT recruits a neuroendocrine signal promoting vasoconstriction. The elevated circulating OXT that followed optogenetic excitation of PVN^OXT was a parsimonious candidate for this neuroendocrine signal; however, the increased SBP persisted after systemic administration of an OTA. In vitro studies revealed that optogenetic excitation of PVN^OXT evokes calcium flux in sniffer cells expressing OTRs or V1aRs. The implication is that firing of PVN^OXT is followed by local release of OXT that binds to OTRs as well as V1aRs. Indeed, we found that PVN^AVP express Fos, a marker of neuronal activation, after the optogenetic excitation of PVN^OXT. Intriguingly, the optogenetic excitation of PVN^OXT also augments firing of PVN^AVP in our ex vivo preparation and tends to increase circulating AVP in vivo. Remarkably, systemic or central administration of V1aR antagonists completely abolished the increased SBP that followed optogenetic excitation of PVN^OXT. Collectively, our results reveal that evoked firing of PVN^OXT leads to the local release of OXT that increases SBP by activating V1aR(s) expressed on PVN^AVP. This, in turn, results in increased circulating AVP and elevated SBP, likely due to systemic vasoconstriction.

Cross Talk Among Neurosecretory Neurons Mediates Cardiovascular Function

Prior studies found that mice deficient in OXT exhibit hypotension and altered baroreflex function.³² In our experiments, optogenetic excitation of PVN^OXT increased SBP in male and female mice. This is in agreement with previous studies suggesting that the release of OXT by the PVN modulates cardiovascular function.¹⁶ Conversely, we found that optogenetic inhibition of PVN^OXT lowered SBP, indicating that PVN^OXT are tonically active and contributes to the maintenance of SBP. These results, in conjunction with our previous work indicating that corticotropin-releasing hormone affects the basal firing rates of PVN preautonomic neurons,¹⁹ provide evidence that paracrine signals originating from hypothalamic neurosecretory neurons regulate SBP. Moreover, the present results suggest that upregulation or downregulation of PVN^OXT activity controls intrahypothalamic release of OXT, which coordinates the secretion of neuroendocrine factors maintaining SBP.

We found that optogenetic excitation of PVN^OXT increases SBP and decreases HR; however, the increased SBP persisted during ganglionic blockade but the decreased HR was abolished. This suggests that autonomic outflow to cardiovascular tissues is not necessary for the increased SBP but is required for the reduced HR that accompanies optogenetic excitation of PVN^OXT. HR can be modulated by PVN^OXT via connections to the hindbrain and spinal cord. For example, parvocellular PVN^OXT have direct projections to sympathetic and parasympathetic preganglionic neurons.^{10,11,14,15,33} Specific projections to the dorsal motor nucleus of the vagus or pre-Bötzinger complex engage parasympathetic efferents that influence HR or respiratory rhythm, respectively.^10,11,33 The selective excitation of these oxytocinergic projections reduces SBP and HR.^12,33 In addition, PVN^OXT has direct excitatory spinal projections to sympathetic preganglionic neurons^14,15 and stimulation of these projections with intrathecal injection of OXT also affects HR.¹⁶ Taking these prior studies into account, it is possible that the bradycardia that we observed results from excitation of parvocellular PVN^OXT that project to the hindbrain or spinal cord. Alternatively, the bradycardia may be the product of neuroendocrine signals that promote vasoconstriction and baroreflex activation. In this regard, AVP, but not OXT, is a major vasoconstrictor neurohormone released by magnocellular neurons, and the pressor response and bradycardia that accompanied optogenetic excitation of PVN^OXT were abolished with a V1aR antagonist. Given that systemic delivery of AVP elicits the baroreflex,³¹ it is likely that the reduced HR that accompanies excitation of PVN^OXT results from AVP-evoked baroreflex activation.

Optogenetic excitation of PVN^OXT elicited increases in SBP that differed in magnitude among male and female mice. Despite implementing similar frequencies for optogenetic stimulation, male mice exhibited a pressor response that was ≈2-fold greater than that of females. Consistent with the blunted pressor response, the bradycardia that we observed was absent in female mice after the optogenetic excitation of PVN^OXT. One possibility is that discrepancies in basal levels of blood pressure may have contributed to this sex difference; however, baseline SBP was similar among males and females, and we did not find significant correlations between baseline SBP and the magnitude of the pressor response. Rather, we propose that the increases in SBP that occurred in females were insufficient to elicit baroreflex activation, and additional studies are needed to address underlying mechanisms.

Mechanisms Underlying Intrahypothalamic Paracrine Signaling

Earlier studies found that PVN^OXT or PVN^AVP each expresses its cognate receptor, enabling autoregulation of their activity and neurohypophysial release.^34,35 However, OXT can bind and act on AVP receptors,^36–41 and in vitro electrophysiological studies revealed that OXT released by PVN^OXT can stimulate V1aR.⁴² Hence, we tested the possibility that the actions of PVN^OXT might be mediated through OXT binding to V1aR on PVN^AVP. We utilized Chinese hamster ovary cells containing human OTRs or V1aRs as a proxy for OXT release and binding to these respective receptors. Consistent with prior reports that OXT binds to both OTRs and V1aRs (albeit with a relatively lesser affinity),^36–41 we found that optogenetic excitation of PVN^OXT increased calcium activity in Chinese hamster ovary cells expressing OTRs as well as V1aRs. These results, in conjunction with our ex vivo electrophysiological recordings demonstrating that excitation of PVN^OXT elicits concurrent firing of neighboring PVN^AVP, predicted in vivo cross talk among these peptidergic neurons. Indeed, we discovered that in vivo optogenetic stimulation of PVN^OXT increases Fos immunoreactivity within PVN^AVP. Several features of this response suggest that a paracrine, diffusible signal mediates the coupling between PVN^OXT and PVN^AVP. First, higher pulses and sustained stimulations were required to observe this coupling. This suggests that high concentrations of OXT were needed to generate a large diffusible signal sufficient to activate relatively distant V1aRs with low affinity for OXT. Second, and perhaps more importantly, the coupling occurred after a relatively long delay (in the order of tens of seconds), and this temporal pattern is indicative of diffusible signals.^18,19,43

Thus, our data suggest that peptidergic interneuronal cross talk between PVN^OXT and PVN^AVP regulates SBP. This notion of paracrine signaling between PVN neuronal subpopulations is an emerging phenomenon. Our group has previously demonstrated an intra-PVN cross talk between neuroendocrine corticotropin-releasing hormone and presympathetic neurons,¹⁹ as well as between PVN^AVP and presympathetic neurons¹⁸ both of which regulate SBP under different physiological conditions. Here, we propose a novel intra-PVN cross talk between PVN^OXT and PVN^AVP. Based on the results from our ex vivo (ie, recordings obtained from identified neurosecretory AVP neurons) and in vivo studies (ie, cross talk increases SBP via systemically acting AVP), we can reasonably conclude that firing of PVN^OXT is coupled to the firing of magnocellular PVN^AVP. Thus, our studies compellingly demonstrate the importance of an interpopulation cross talk between PVN^OXT and PVN^AVP in the regulation of systemic SBP.

Limitations

Initially, we revealed that low-frequency (5 Hz) optogenetic excitation of PVN^OXT in conscious mice produced a pressor response (>10 mm Hg). We also found that excitation of PVN^OXT in conscious mice elicited behavioral responses (eg, grooming, yawning). The behaviors accompanying excitation of PVN^OXT were consistent with prior studies^44,45 and may contribute to the cardiovascular responses that we observed. Accordingly, we used our anesthetized in vivo optogenetic preparation to isolate the cardiovascular effects and found that stimulation of PVN^OXT at 30 Hz reliably produced pressor responses. Anesthesia suppresses brain activity and the neural connections that mediate cardiovascular function.⁴⁶ Although we acknowledge that assessing cardiovascular parameters under anesthesia is a limitation of our study, it is important to note that the rates of neuronal discharge that were used to evoke pressor responses are not supraphysiological. Under basal conditions, OXT neurons fire spontaneously at of 1 to 3 Hz⁴⁷ but parturition and uterine contraction are preceded by synchronous firing at 20 to 40 Hz.⁴⁸ There are additional reports of OXT neurons exhibiting high-frequency bursts reaching up to 80 Hz.⁴⁹ In fact, during suckling, OXT neurons exhibit burst firing at 50 to 150 spikes in 1 to 3 seconds,⁴⁷ and during lactation, OXT neurons can fire up to ≈50 Hz.⁴⁸ Therefore, the firing frequencies that we used to evoke pressor responses are within the neurophysiological range for OXT neurons.

Potential Clinical Implications

The coupling between PVN^OXT and PVN^AVP may have pathophysiological implications, particularly during preeclampsia, a condition characterized by elevated SBP⁵⁰ and plasma AVP.⁵¹ A growing body of evidence supports AVP as a reliable and early predictive marker for the development of preeclampsia.^51–53 In this sense, growing evidence indicates that the activity of both OXT and AVP magnocellular neurons increases during the reproductive cycle, particularly during parturition,^54–58 resulting in the enhanced local release of OXT and AVP within the supraoptic nucleus and PVN.^59,60 These studies support the notion that coupling the excitation of PVN^OXT and PVN^AVP could become exaggerated during parturition, and that under pathological conditions, it could act as a contributing factor to abnormally elevated AVP levels and increased SBP, characteristic of preeclampsia. Future studies to test these significant and clinically relevant hypotheses are warranted.

Article Information

Sources of Funding

Supported by American Heart Association (AHA) 23POST1020034 and NHLBI K99HL175100, R00HL175100 to K. Elsaafien, National Heart, Lung, and Blood Institute (NHLBI) F31HL62540 to C. Baumer-Harrison, NHLBI R01HL136595, R35HL150750, R01HL145028 to A. de Kloet and E.G. Krause, NHLBI K99HL168434, R00HL168434 to M.K. Kirchner, National Center for Complementary and Integrative Health (NCCIH) R01AT013166 to A. de Kloet and E.G. Krause.

Disclosures

None.

Supplemental Material

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Tables S1–S7

Figures S1–S5

Video S1

Major Resources Table

References 61–68

ARRIVE Guidelines

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