Oxytocin and Vasopressin Cross Talk Within the Brain Increases Blood Pressure

Figures

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  Figure 1.. Genetic targeting of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT). 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 PVNOXT 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,…

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  Figure 2.. Optogenetic excitation of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT) 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).

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  Figure 3.. Optogenetic inhibition of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT) 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 PVNOXT. 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…

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  Figure 4.. Pressor responses to optogenetically activating OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT) 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.

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  Figure 5.. Pressor responses to optogenetically activating OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT) 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;…

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  Figure 6.. Optogenetic excitation of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT) 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 PVNOXT (left), CHO sniffer cells containing human oxytocin receptors (SnifferOTR) R-GECO signal (middle), and a merged image (right). B, Pseudo-color images of SnifferOTR before (left) and after (right) 488 nm light illumination. Arrowheads mark cells that responded to optogenetic excitation of PVNOXT. C, Representative F/F0 calcium traces corresponding to the arrowheads in B, demonstrating SnifferOTR responses to PVNOXT activation. D, Pie charts summarizing response rates of SnifferOTR (left; n=6) and SnifferV1aR (CHO sniffer cells containing human V1aRs; middle; n=3 animals, 127 cells) plated in paraventricular nucleus of the hypothalamus (PVN), and SnifferOTR plated in cortex (right; n=4 animals, 61 cells), after 488 nm light illumination. E, Quantitative analysis of SnifferOTR responses in the PVN following blue light stimulation. OTR indicates oxytocin receptors; and R-GECO, Red…

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  Figure 7.. Stimulation of oxytocin-synthesizing neurons of the paraventricular nucleus (PVNOXT) 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 PVNOXT. 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…

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  Figure 8.. Optogenetic excitation of OXT (oxytocin)-synthesizing neurons of the paraventricular nucleus (PVNOXT) evokes cross talk with arginine vasopressin-synthesizing neurons of the paraventricular nucleus (PVNAVP) 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…