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 hr normoxia (21% O, n=5) or hypoxia (10% O, 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 CRFR2 were co-localized with oxytocin fibers and were closely associated with hypoxia-activated nTS neurons. A separate group of animals that received microinjection of AAV2-hSyn-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 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.

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The nucleus tractus solitarii (nTS) is the initial central termination site for visceral afferents and is important for modulation and integration of multiple reflexes including cardiorespiratory reflexes. Glutamate is the primary excitatory neurotransmitter in the nTS and is removed from the extracellular milieu by excitatory amino acid transporters (EAATs). The goal of this study was to elucidate the role of EAATs in the nTS on basal synaptic and neuronal function and cardiorespiratory regulation. The majority of glutamate clearance in the central nervous system is believed to be mediated by astrocytic EAAT 1 and 2. We confirmed the presence of EAAT 1 and 2 within the nTS and their colocalization with astrocytic markers. EAAT blockade withdl-threo-β-benzyloxyaspartic acid (TBOA) produced a concentration-related depolarization, increased spontaneous excitatory postsynaptic current (EPSC) frequency, and enhanced action potential discharge in nTS neurons. Solitary tract-evoked EPSCs were significantly reduced by EAAT blockade. Microinjection of TBOA into the nTS of anesthetized rats induced apneic, sympathoinhibitory, depressor, and bradycardic responses. These effects mimicked the response to microinjection of exogenous glutamate, and glutamate responses were enhanced by EAAT blockade. Together these data indicate that EAATs tonically restrain nTS excitability to modulate cardiorespiratory function.

King TL, Ruyle BC, Kline DD, Heesch CM, Hasser EM. Catecholaminergic neurons projecting to the paraventricular nucleus of the hypothalamus are essential for cardiorespiratory adjustments to hypoxia. Am J Physiol Regul Integr Comp Physiol 309: R721-R731, 2015. First published July 8, 2015 doi:10.1152/ajpregu.00540.2014.-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 ϭ 6) or anti-dopamine ␤-hydroxylase saporin (DSAP; n ϭ 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% O 2) and hypercapnia (5% CO 2-95% O2). DSAP decreased the number of tyrosine hydroxylase-immunoreactive terminals in PVN and cells counted in ventrolateral medulla (VLM; Ϫ37%) and nucleus tractus solitarii (nTS; Ϫ36%). 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 (V E) index also were impaired at higher hypoxic intensities (10-8% O 2; e.g., VE at 8% O2: IgG ϭ 181 Ϯ 22, DSAP ϭ 91 Ϯ 4 arbitrary units). Depressed ventilation in DSAP rats was associated with significantly lower arterial O 2 saturation at all hypoxic intensities. PVN DSAP also reduced ventilatory responses to 5% CO 2 (VE: IgG ϭ 176 Ϯ 21 and DSAP ϭ 84 Ϯ 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.chemoreflex; blood pressure; ventilation; anti-dopamine ␤-hydroxylase saporin; brainstem PERIPHERAL CHEMOREFLEX ACTIVATION by systemic hypoxia produces highly integrated respiratory, autonomic, behavioral, and endocrine responses (15,17). Together these responses are critical for homeostasis, serving to restore and maintain tissue oxygenation. The central nervous system pathways involved in responses to acute hypoxia are complex. Peripheral chemoreceptor afferent nerves from the carotid bodies project onto neurons located in the nucleus tractus solitarii (nTS) (40, 51), where chemoreceptor afferent input is modulated and integrated. The nTS sends projections to the rostral ventrolateral medulla (RVLM) (28,30), and this projection is considered the primary pathway mediating cardiorespiratory chemoreflex responses.The hypothalamic paraventricular nucleus (PVN) is also important in the integrated respo...

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% O2) 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% O2, 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 PVN contributes to cardiorespiratory responses to peripheral chemoreflex activation. We have shown that hypoxia (Hx) activates PVN neurons that project to the nTS but the functional role of this nTS projection is not known. We hypothesized that selective inhibition/activation of the PVN to nTS pathway blunts/augments chemoreflex cardiorespiratory responses. Male SD rats received bilateral microinjections of retrograde AAV‐pgk‐Cre into the nTS. One week later, rats received bilateral PVN injections of Cre‐dependent AAV2‐DIO‐hSyn‐mCherry expressing inhibitory (Gi) or excitatory (Gq) DREADD, or control virus (mCh), and 3–5 weeks allowed for expression in nTS‐projecting PVN neurons. To evaluate the contribution of the PVN to nTS pathway to chemoreflex function, ventilatory responses (plethysmography) to progressive Hx (14–8% O2) were assessed in conscious animals before and after ip injection of saline or the synthetic selective DREADD ligand C21 (1mg/kg). We similarly evaluated Hx‐induced nTS neuronal activation (Fos immunohistochemistry); 60 min after ip saline or C21, conscious mCh (n=6) and Gi (n=4) rats were exposed to Hx (2 hr, 10% O2) and Gq rats were exposed to 12% O2. Saline had no effect on either ventilatory responses or nTS neuronal activation to Hx in any group. In Gi rats, selective inhibition of nTS‐projecting PVN neurons with C21 blunted hypoxic ventilatory responses to 10% and 8% O2 (n=8, p<0.05) and appeared associated with decreased Hx‐induced nTS neuronal activation (581 ± 33 vs. 375 ± 150 cells; saline vs. C21; n=2 each). In contrast, selective activation of the PVN to nTS pathway by C21 in Gq rats (n=8) enhanced Hx ventilatory responses to milder hypoxia (14 and 12% O2, p<0.05), and produced greater Hx‐induced Fos‐IR in the nTS (377 ± 45 vs. 550 ± 23 cells; saline vs. C21, n=3 ea). To confirm that altered cardiorespiratory chemoreflex responses were mediated by PVN terminals in the nTS, anesthetized rats were exposed to brief (45s) Hx episodes (10% O2, mCh and Gi rats; 12% O2, Gq rats) before and after bilateral nTS microinjection of C21 (0.1mM; 90nl/side). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and phrenic nerve activity (PhrNA)] were measured. Under control conditions, Hx decreased MAP and increased HR, sSNA and PhrNA in all groups. Peak responses to 10% O2 were similar in mCh and Gi rats and greater than control responses to 12% O2 in Gq rats. nTS microinjection of C21 had minor baseline effects, which were not different among groups. nTS C21 did not alter the Hx‐induced increase in PhrNA of mCh rats (n=2). In Gi rats (n=3), DREADD‐mediated inhibition of PVN terminals in the nTS blunted both PhrNA (−63 ± 10%; p<0.05) and sSNA (−32 ± 18%; p<0.05) responses to Hx. In contrast, activation of PVN terminals in the nTS enhanced both PhrNA (+51 ± 36%) and sSNA (+166 ± 41%) responses to Hx in Gq rats (n=3). Together, these results suggest that a PVN to nTS pathway directly enhances nTS neuronal activation and cardiorespiratory responses to hypoxia.Support or Funding InformationRO1‐HL‐98602 F31‐HL‐140858This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The hypothalamic paraventricular nucleus (PVN) contributes to cardiorespiratory responses to peripheral chemoreflex activation. Our previous data indicate that acute hypoxia (Hx) activates PVN neuropeptidergic neurons that project to the nucleus tractus solitarii (nTS), including corticotropin releasing hormone and oxytocin (OT) neurons. However, the functional role of these projections to the nTS during Hx has not been fully delineated. We hypothesized PVN inputs to the nTS facilitate cardiorespiratory responses to Hx, and this requires activation of neuropeptidergic, including OT, receptors. Male Sprague Dawley rats received bilateral microinjections of AAV‐hSyn‐Gi‐DREADD‐mCherry (inhibitory DREADD) or AAV‐hSyn‐GFP (control) into the PVN. 3–5 weeks were allowed for expression in PVN neurons and in their projections. Rats were anesthetized, and cardiorespiratory parameters [blood pressure, (BP), heart rate (HR) splanchnic sympathetic nerve activity (sSNA) and phrenic nerve activity (PhrNA)] were measured at baseline and during Hx (10% O2, 45s). Gi‐DREADD‐expressing PVN terminals specifically in the nTS were inhibited via bilateral nTS microinjection of a synthetic selective DREADD ligand, Compound 21 (C21, 0.1mM; 90nl/side). Cardiorespiratory responses to Hx were measured before and 1 hr after DREADD‐mediated inhibition of PVN terminals in the nTS. After the experiment, brains were removed, and immunohistochemistry was performed on PVN and nTS tissue to verify AAV injection sites in the PVN and terminal expression in the nTS. We observed robust expression of mCherry and GFP containing cells in the PVN and dense fibers throughout the nTS in Gi‐DREADD and GFP rats, respectively. A portion of these fibers also were OT immunoreactive. Under control conditions, Hx significantly decreased BP and increased HR, sSNA and PhrNA frequency, amplitude (PhrAmp) and minute activity (MinPhrNA). Responses were similar in GFP and Gi‐DREADD rats. nTS microinjection of C21 had minor effects on baseline cardiorespiratory parameters, which were not significantly different between the two groups. In Gi‐DREADD rats, inhibition of PVN terminals in the nTS (C21 microinjection) decreased the MinPhrNA response to Hx by 35±13% (p<0.05, n=6). The effect was primarily due to reduced PhrAmp (41±7%, p<0.07, n=6). In contrast, the Hx‐induced increase in MinPhrNA was not significantly affected by C21 in the nTS of GFP rats (% increase MinPhrNA, Ctrl Hx, 425±13; Hx 60’ post‐C21, 535±142, n=3). Comparison between groups indicated that PhrNA during Hx 1 hr post‐C21 was significantly less in Gi‐DREADD vs GFP rats. To evaluate a role for OT in the nTS, in separate animals (n=2) the OT receptor antagonist OTA (0.1mM) was bilaterally microinjected in the nTS. OTA blunted the Hx‐induced increase in MinPhrNA (mV.s: Ctrl Hx, 78±16; Hx post‐OTA, 45±20). Thus, inhibition of PVN terminals and OT receptors in the nTS both blunted Hx‐evoked chemoreflex cardiorespiratory output. Together, these results are consistent with the concept that PVN inputs to the nTS are required for full expression of cardiorespiratory chemoreflex responses, and OTergic mechanisms in the nTS may contribute.Support or Funding InformationNIH RO1 HL98602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.