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Chemoreflex neurocircuitry includes the paraventricular nucleus (PVN), but the role of PVN efferent projections to specific cardiorespiratory nuclei is unclear. We hypothesized that the PVN contributes to cardiorespiratory responses to hypoxia via projections to the nucleus tractus solitarii (nTS). Rats received bilateral PVN microinjections of adeno-associated virus expressing inhibitory designer receptor exclusively activated by designer drug (GiDREADD) or green fluorescent protein (GFP) control. Efficacy of GiDREADD inhibition by the designer receptor exclusively activated by designer drug (DREADD) agonist Compound 21 (C21) was verified in PVN slices; C21 reduced evoked action potential discharge by reducing excitability to injected current in GiDREADD-expressing PVN neurons. We evaluated hypoxic ventilatory responses (plethysmography) and PVN and nTS neuronal activation (cFos immunoreactivity) to 2 h hypoxia (10% O 2 ) in conscious GFP and GiDREADD rats after intraperitoneal C21 injection. Generalized PVN inhibition via systemic C21 blunted hypoxic ventilatory responses and reduced PVN and also nTS neuronal activation during hypoxia. To determine if the PVN-nTS pathway contributes to these effects, we evaluated cardiorespiratory responses to hypoxia during selective PVN terminal inhibition in the nTS. Anesthetized GFP and GiDREADD rats exposed to brief hypoxia (10% O 2 , 45 s) exhibited depressor and tachycardic responses and increased sympathetic and phrenic nerve activity. C21 was then microinjected into the nTS, followed after 60 min by another hypoxic episode. In GiDREADD but not GFP rats, PVN terminal inhibition by nTS C21 strongly attenuated the phrenic amplitude response to hypoxia. Interestingly, C21 augmented tachycardic and sympathetic responses without altering the coupling of splanchnic sympathetic nerve activity to phrenic nerve activity during hypoxia. Data demonstrate that the PVN, including projections to the nTS, is critical in shaping sympathetic and respiratory responses to hypoxia.

The paraventricular nucleus of the hypothalamus (PVN) contributes to both autonomic and neuroendocrine function. PVN lesion or inhibition blunts cardiorespiratory responses to peripheral chemoreflex activation, suggesting that the PVN is required for full expression of these effects. However, the role of efferent projections to cardiorespiratory nuclei and the neurotransmitters/neuromodulators that are involved is unclear. The PVN sends dense projections to the nucleus tractus solitarii (nTS), a region that displays neuronal activation following hypoxia. We hypothesized that acute hypoxia activates nTS-projecting PVN neurons. Using a combination of retrograde tracing and immunohistochemistry, we determined whether hypoxia activates PVN neurons that project to the nTS and examined the phenotype of these neurons. Conscious rats underwent 2 h normoxia (21% O 2 , n = 5) or hypoxia (10% O 2 , n = 6). Hypoxia significantly increased Fos immunoreactivity in nTS-projecting neurons, primarily in the caudal PVN. The majority of activated nTS-projecting neurons contained corticotropin-releasing hormone (CRH). In the nTS, fibers expressing the CRH receptor corticotropin-releasing factor receptor 2 (CRFR2) were colocalized with oxytocin (OT) fibers and were closely associated with hypoxia-activated nTS neurons. A separate group of animals that received a microinjection of adeno-associated virus type 2-hSyn-green fluorescent protein (GFP) into the PVN exhibited GFP-expressing fibers in the nTS; a proportion of these fibers displayed OT immunoreactivity. Thus, nTS CRFR2s appear to be located on the fibers of PVN OT neurons that project to the nTS. Taken together, our findings suggest that PVN CRH projections to the nTS may modulate nTS neuronal activation, possibly via OTergic mechanisms, and thus contribute to chemoreflex cardiorespiratory responses.

Hypoxia results in decreased arterial Po 2 , arterial chemoreflex activation, and compensatory increases in breathing, sympathetic outflow, and neuroendocrine secretions, including increased secretion of AVP, corticotropin-releasing hormone (CRH), adrenocorticotropin hormone (ACTH), and corticosterone. In addition to a brain stem pathway, including the nucleus tractus solitarius (nTS) and the rostral ventrolateral medulla (RVLM), medullary pathways to the paraventricular nucleus of the hypothalamus (PVN) contribute to chemoreflex responses. Experiments evaluated activation of specific cell phenotypes within the PVN following an acute hypoxic stimulus (AH; 2 h, 10% O 2 ) in conscious rats. Retrograde tracers (from spinal cord and RVLM) labeled presympathetic (PreS) neurons, and immunohistochemistry identified AVP- and CRH-immunoreactive (IR) cells. c-Fos-IR was an index of neuronal activation. Hypoxia activated AVP-IR (~6%) and CRH-IR (~15%) cells, but not PreS cells in the PVN, suggesting that sympathoexcitation during moderate AH is mediated mainly by a pathway that does not include PreS neurons in the PVN. Approximately 14 to 17% of all PVN cell phenotypes examined expressed neuronal nitric oxide synthase (nNOS-IR). AH activated only nNOS-negative AVP-IR neurons. In contrast ~23% of activated CRH-IR neurons in the PVN contained nNOS. In the median eminence, CRH-IR terminals were closely opposed to tanycyte processes and end-feet (vimentin-IR) in the external zone, where vascular NO participates in tanycyte retraction to facilitate neuropeptide secretion into the pituitary portal circulation. Results are consistent with an inhibitory role of NO on AVP and PreS neurons in the PVN and an excitatory role of NO on CRH secretion in the PVN and median eminence., (Copyright © 2017 the American Physiological Society.)

Pregnancy is characterized by increased blood volume and baseline sympathetic nerve activity (SNA), vasodilation, and tachycardia. Relaxin (RLX), an ovarian hormone elevated in pregnancy, activates forebrain sites involved in control of blood volume and SNA through ANG II-dependent mechanisms and contributes to adaptations during pregnancy. In anesthetized, arterial baroreceptor-denervated nonpregnant (NP) rats, RLX microinjected into the subfornical organ (SFO; 0.77 pmol in 50 nl) produced sustained increases in lumbar SNA (8 ± 3%) and mean arterial pressure (MAP; 26 ± 4 mmHg). Low-dose intracarotid artery infusion of RLX (155 pmol·ml(-1)·h(-1); 1.5 h) had minor transient effects on AP and activated neurons [increased Fos-immunoreactivity (IR)] in the SFO and in spinally projecting (19 ± 2%) and arginine-vasopressin (AVP)-IR (21 ± 5%) cells in the paraventricular nucleus of the hypothalamus of NP, but not pregnant (P), rats. However, mRNA for RLX and ANG II type 1a receptors in the SFO was preserved in pregnancy. RLX receptor-IR is present in the region of the SFO in NP and P rats and is localized in astrocytes, the major source of angiotensinogen in the SFO. These data provide an anatomical substrate for a role of RLX in the resetting of AVP secretion and increased baseline SNA in pregnancy. Since RLX and ANG II receptor expression was preserved in the SFO of P rats, we speculate that the lack of response to exogenous RLX may be due to maximal activation by elevated endogenous levels of RLX in near-term pregnancy., (Copyright © 2015 the American Physiological Society.)

Brainstem catecholamine neurons modulate sensory information and participate in control of cardiorespiratory function. These neurons have multiple projections, including to the paraventricular nucleus (PVN), which contributes to cardiorespiratory and neuroendocrine responses to hypoxia. We have shown that PVN-projecting catecholaminergic neurons are activated by hypoxia, but the function of these neurons is not known. To test the hypothesis that PVN-projecting catecholamine neurons participate in responses to respiratory challenges, we injected IgG saporin (control; n = 6) or anti-dopamine β-hydroxylase saporin (DSAP; n = 6) into the PVN to retrogradely lesion catecholamine neurons projecting to the PVN. After 2 wk, respiratory measurements (plethysmography) were made in awake rats during normoxia, increasing intensities of hypoxia (12, 10, and 8% O2) and hypercapnia (5% CO2-95% O2). DSAP decreased the number of tyrosine hydroxylase-immunoreactive terminals in PVN and cells counted in ventrolateral medulla (VLM; -37%) and nucleus tractus solitarii (nTS; -36%). DSAP produced a small but significant decrease in respiratory rate at baseline (during normoxia) and at all intensities of hypoxia. Tidal volume and minute ventilation (VE) index also were impaired at higher hypoxic intensities (10-8% O2; e.g., VE at 8% O2: IgG = 181 ± 22, DSAP = 91 ± 4 arbitrary units). Depressed ventilation in DSAP rats was associated with significantly lower arterial O2 saturation at all hypoxic intensities. PVN DSAP also reduced ventilatory responses to 5% CO2 (VE: IgG = 176 ± 21 and DSAP = 84 ± 5 arbitrary units). Data indicate that catecholamine neurons projecting to the PVN are important for peripheral and central chemoreflex respiratory responses and for maintenance of arterial oxygen levels during hypoxic stimuli., (Copyright © 2015 the American Physiological Society.)

Hypoxia activates catecholamine neurons in the caudal ventrolateral medulla (CVLM). The hypothalamic paraventricular nucleus (PVN) modulates arterial chemoreflex responses and receives catecholaminergic projections from the CVLM, but it is not known whether the CVLM-PVN projection is activated by chemoreflex stimulation. We hypothesized that acute hypoxia (AH) activates PVN-projecting catecholaminergic neurons in the CVLM. Fluoro-Gold (2%, 60-90 nl) was microinjected into the PVN of rats to retrogradely label CVLM neurons. After recovery, conscious rats underwent 3 h of normoxia (21% O2, n = 4) or AH (12, 10, or 8% O2; n = 5 each group). We used Fos immunoreactivity as an index of CVLM neuronal activation and tyrosine hydroxylase (TH) immunoreactivity to identify catecholaminergic neurons. Positively labeled neurons were counted in six caudal-rostral sections containing CVLM. Hypoxia progressively increased the number of Fos-immunoreactive CVLM cells (21%, 19 ± 6; 12%, 49 ± 2; 10%, 117 ± 8; 8%, 179 ± 7; P < 0.001). Catecholaminergic cells colabeled with Fos immunoreactivity in the CVLM were observed following 12% O2, and further increases in hypoxia severity caused markedly more activation. PVN-projecting CVLM cells were activated following more severe hypoxia (10% and 8% O2). A large proportion (89 ± 3%) of all activated PVN-projecting CVLM neurons were catecholaminergic, regardless of hypoxia intensity. Data suggest that catecholaminergic, PVN-projecting CVLM neurons are particularly hypoxia-sensitive, and these neurons may be important in the cardiorespiratory and/or neuroendocrine responses elicited by the chemoreflex.

Peripheral chemoreceptor afferent information is sent to the nucleus tractus solitarii (nTS), integrated, and relayed to other brain regions to alter cardiorespiratory function. The nTS projects to the hypothalamic paraventricular nucleus (PVN), but activation and phenotype of these projections during chemoreflex stimulation is unknown. We hypothesized that activation of PVN-projecting nTS neurons occurs primarily at high intensities of hypoxia. We assessed ventilation and cardiovascular parameters in response to increasing severities of hypoxia. Retrograde tracers were used to label nTS PVN-projecting neurons and, in some rats, rostral ventrolateral medulla (RVLM)-projecting neurons. Immunohistochemistry was performed to identify nTS cells that were activated (Fos-immunoreactive, Fos-IR), catecholaminergic, and GABAergic following hypoxia. Conscious rats underwent 3 h normoxia (n = 4, 21% O(2)) or acute hypoxia (12, 10, or 8% O(2); n = 5 each). Hypoxia increased ventilation and the number of Fos-IR nTS cells (21%, 13 ± 2; 12%, 58 ± 4; 10%, 166 ± 22; 8%, 186 ± 6). Fos expression after 10% O(2) was similar whether arterial pressure was allowed to decrease (-13 ± 1 mmHg) or was held constant. The percentage of PVN-projecting cells activated was intensity dependent, but contrary to our hypothesis, PVN-projecting nTS cells exhibiting Fos-IR were found at all hypoxic intensities. Notably, at all intensities of hypoxia, ∼75% of the activated PVN-projecting nTS neurons were catecholaminergic. Compared with RVLM-projecting cells, a greater percentage of PVN-projecting nTS cells was activated by 10% O(2). Data suggest that increasing hypoxic intensity activates nTS PVN-projecting cells, especially catecholaminergic, PVN-projecting neurons. The nTS to PVN catecholaminergic pathway may be critical even at lower levels of chemoreflex activation and more important to cardiorespiratory responses than previously considered.

The purpose of this review is to delineate the general features of endocrine regulation of the baroreceptor reflex, as well as specific contributions during pregnancy. In contrast to the programmed changes in baroreflex function that occur in situations initiated by central command (e.g., exercise or stress), the complex endocrine milieu often associated with physiological and pathophysiological states can influence the central baroreflex neuronal circuitry via multiple sites and mechanisms, thereby producing varied changes in baroreflex function. During pregnancy, baroreflex gain is markedly attenuated, and at least two hormonal mechanisms contribute, each at different brain sites: increased levels of the neurosteroid 3alpha-hydroxy-dihydroprogesterone (3alpha-OH-DHP), acting in the rostral ventrolateral medulla (RVLM), and reduced actions of insulin in the forebrain. 3alpha-OH-DHP appears to potentiate baroreflex-independent GABAergic inhibition of premotor neurons in the RVLM, which decreases the range of sympathetic nerve activity that can be elicited by changes in arterial pressure. In contrast, reductions in the levels or actions of insulin in the brain blunt baroreflex efferent responses to increments or decrements in arterial pressure. Although plasma levels of angiotensin II are increased in pregnancy, this is not responsible for the reduction in baroreflex gain, although it may contribute to the increased level of sympathetic nerve activity in this condition. How these different hormonal effects are integrated within the brain, as well as possible interactions with additional potential neuromodulators that influence baroreflex function during pregnancy and other physiological and pathophysiological states, remains to be clearly delineated.

During baroreceptor unloading, sympathoexcitation is attenuated in near-term pregnant compared with nonpregnant rats. Alterations in balance among different excitatory and inhibitory inputs within central autonomic pathways likely contribute to changes in regulation of sympathetic outflow in pregnancy. Both baroreflex-dependent and baroreflex-independent GABAergic inputs inhibit sympathoexcitatory neurons within rostral ventrolateral medulla (RVLM). The present experiments tested the hypothesis that influence of baroreflex-independent GABAergic inhibition of RVLM is greater in pregnant compared with nonpregnant rats. Afferent baroreceptor inputs were eliminated by bilateral sinoaortic denervation in inactin-anesthetized rats. In pregnant compared with nonpregnant rats, baseline mean arterial pressure (MAP) was lower (pregnant = 75 +/- 6 mmHg, nonpregnant = 115 +/- 7 mmHg) and heart rate was higher (pregnant = 381 +/- 10 beats/min, nonpregnant = 308 +/- 10 beats/min). Pressor and sympathoexcitatory [renal sympathetic nerve activity, (RSNA)] responses due to bilateral GABA(A) receptor blockade (bicuculline, 4 mM, 100 nl) of the RVLM were greater in pregnant rats (delta MAP: pregnant = 101 +/- 4 mmHg, nonpregnant = 80 +/- 6 mmHg; delta RSNA: pregnant = 182 +/- 23% control, nonpregnant = 133 +/- 10% control). Unexpected transient sympathoexcitatory effects of angiotensin AT(1) receptor blockade in the RVLM were greater in pregnant rats. Although excitatory responses to bicuculline were attenuated by prior RVLM AT1 receptor blockade in both groups, pressor responses to disinhibition of the RVLM remained augmented in pregnant rats. Increased influence of baroreflex-independent GABAergic inhibition in RVLM could contribute to suppressed sympathoexcitation during withdrawal of arterial baroreceptor input in pregnant animals.

Exposure to a period of microgravity or bed rest produces several physiological adaptations. These changes, which include an increased incidence of orthostatic intolerance, have an impact when people return to a 1G environment or resume an upright posture. Compared with males, females appear more susceptible to orthostatic intolerance after exposure to real or simulated microgravity. Decreased arterial baroreflex compensation may contribute to orthostatic intolerance. We hypothesized that female rats would exhibit a greater reduction in arterial baroreflex function after hindlimb unloading (HU) compared with male rats. Mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in conscious animals after 13-15 days of HU. Baseline HR was elevated in female rats, and HU increased HR in both genders. Consistent with previous results in males, baroreflex-mediated activation of RSNA was blunted by HU in both genders. Maximum RSNA in response to decreases in MAP was reduced by HU (male control 513 +/- 42%, n = 11; male HU 346 +/- 38%, n = 13; female control 359 +/- 44%, n = 10; female HU 260 +/- 43%, n = 10). Maximum baroreflex increase in RSNA was lower in females compared with males in both control and HU rats. Both female gender and HU attenuated baroreflex-mediated increases in sympathetic activity. The combined effects of HU and gender resulted in reduced baroreflex sympathetic reserve in females compared with males and could contribute to the greater incidence of orthostatic intolerance in females after exposure to spaceflight or bed rest.

Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.