Orexin facilitates the peripheral chemoreflex via corticotropin releasing hormone neurons projecting to the nucleus of the solitary tract.

We previously showed that orexin neurons are activated by hypoxia and facilitate the peripheral chemoreflex (PCR)-mediated hypoxic ventilatory response (HVR), mostly by promoting the respiratory frequency response. Orexin neurons project to the nucleus of the solitary tract (nTS) and the paraventricular nucleus of the hypothalamus (PVN). The PVN contributes significantly to the PCR and contains nTS-projecting corticotropin releasing hormone (CRH) neurons. We hypothesized that in male rats orexin neurons contribute to the PCR by activating nTS-projecting CRH neurons. We used neuronal tract tracing and immunohistochemistry (IHC) to quantify the degree that hypoxia activates PVN-projecting orexin neurons. We coupled this with orexin receptor (OxR) blockade with suvorexant (Suvo, 20 mg/kg, i.p.) to assess the degree that orexin facilitates the hypoxia-induced activation of CRH neurons in the PVN, including those projecting to the nTS. In separate groups of rats, we measured the PCR following systemic orexin 1 receptor (Ox1R) blockade (SB-334867;1 mg/kg) and specific Ox1R knockdown in PVN. OxR blockade with Suvo reduced the number of nTS and PVN neurons activated by hypoxia, including those CRH neurons projecting to nTS. Hypoxia increased the number of activated PVN-projecting orexin neurons but had no effect on the number of activated nTS-projecting orexin neurons. Global Ox1R blockade and partial Ox1R knockdown in the PVN significantly reduced the PCR. Ox1R knockdown also reduced the number of activated PVN neurons and the number of activated tyrosine-hydroxylase neurons in the nTS. Our findings suggest orexin facilitates the PCR via nTS-projecting CRH neurons expressing Ox1R.Significance Statement Previously we showed that orexin contributes to the peripheral chemoreflex (PCR), but the mechanisms underlying this effect remain unknown. Here we show that: 1) orexin receptor blockade reduces the activation of the PVN and nTS; 2) hypoxia activates orexin neurons that project to the PVN, but not those projecting to nTS; 3) orexin receptor blockade reduces the activation of nTS-projecting corticotrophin releasing hormone (CRH) neurons in the PVN; 4) orexin 1 receptor (Ox1R) blockade and specific Ox1R knockdown in the PVN reduce the strength of the PCR, and 5) Ox1R knockdown reduces the number of activated PVN neurons and tyrosine-hydroxylase neurons in the nTS. These findings suggest that PVN-projecting orexin neurons facilitate the PCR via Ox1R on nTS-projecting CRH neurons.

Oxytocin and corticotropin-releasing hormone exaggerate nucleus tractus solitarii neuronal and synaptic activity following chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) in rodents mimics the hypoxia-induced elevation of blood pressure seen in individuals experiencing episodic breathing. The brainstem nucleus tractus solitarii (nTS) is the first site of visceral sensory afferent integration, and thus is critical for cardiorespiratory homeostasis and its adaptation during a variety of stressors. In addition, the paraventricular nucleus of the hypothalamus (PVN), in part through its nTS projections that contain oxytocin (OT) and/or corticotropin-releasing hormone (CRH), contributes to cardiorespiratory regulation. Within the nTS, these PVN-derived neuropeptides alter nTS activity and the cardiorespiratory response to hypoxia. Nevertheless, their contribution to nTS activity after CIH is not fully understood. We hypothesized that OT and CRH would increase nTS activity to a greater extent following CIH, and co-activation of OT+CRH receptors would further magnify nTS activity. Our data show that compared to their normoxic controls, 10 days' CIH exaggerated nTS discharge, excitatory synaptic currents and Ca2+ influx in response to CRH, which were further enhanced by the addition of OT. CIH increased the tonic functional contribution of CRH receptors, which occurred with elevation of mRNA and protein. Together, our data demonstrate that intermittent hypoxia exaggerates the expression and function of neuropeptides on nTS activity. KEY POINTS: Episodic breathing and chronic intermittent hypoxia (CIH) are associated with autonomic dysregulation, including elevated sympathetic nervous system activity. Altered nucleus tractus solitarii (nTS) activity contributes to this response. Neurons originating in the paraventricular nucleus (PVN), including those containing oxytocin (OT) and corticotropin-releasing hormone (CRH), project to the nTS, and modulate the cardiorespiratory system. Their role in CIH is unknown. In this study, we focused on OT and CRH individually and together on nTS activity from rats exposed to either CIH or normoxia control. We show that after CIH, CRH alone and with OT increased to a greater extent overall nTS discharge, neuronal calcium influx, synaptic transmission to second-order nTS neurons, and OT and CRH receptor expression. These results provide insights into the underlying circuits and mechanisms contributing to autonomic dysfunction during periods of episodic breathing.

Vagotomy blunts cardiorespiratory responses to vagal afferent stimulation via pre- and postsynaptic effects in the nucleus tractus solitarii.

Viscerosensory information travels to the brain via vagal afferents, where it is first integrated within the brainstem nucleus tractus solitarii (nTS), a critical contributor to cardiorespiratory function and site of neuroplasticity. We have shown that decreasing input to the nTS via unilateral vagus nerve transection (vagotomy) induces morphological changes in nTS glia and reduces sighs during hypoxia. The mechanisms behind post-vagotomy changes are not well understood. We hypothesized that chronic vagotomy alters cardiorespiratory responses to vagal afferent stimulation via blunted nTS neuronal activity. Male Sprague-Dawley rats (6 weeks old) underwent right cervical vagotomy caudal to the nodose ganglion, or sham surgery. After 1 week, rats were anaesthetized, ventilated and instrumented to measure mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively). Vagal afferent stimulation (2-50 Hz) decreased cardiorespiratory parameters and increased neuronal Ca2+ measured by in vivo photometry and in vitro slice imaging of nTS GCaMP8m. Vagotomy attenuated both these reflex and neuronal Ca2+ responses compared to shams. Vagotomy also reduced presynaptic Ca2+ responses to stimulation (Cal-520 imaging) in the nTS slice. The decrease in HR, SSNA and PhrNA due to nTS nanoinjection of exogenous glutamate also was tempered following vagotomy. This effect was not restored by blocking excitatory amino acid transporters. However, the blunted responses were mimicked by NMDA, not AMPA, nanoinjection and were associated with reduced NR1 subunits in the nTS. Altogether, these results demonstrate that vagotomy induces multiple changes within the nTS tripartite synapse that influence cardiorespiratory reflex responses to afferent stimulation. KEY POINTS: Multiple mechanisms within the nucleus tractus solitarii (nTS) contribute to functional changes following vagal nerve transection. Vagotomy results in reduced cardiorespiratory reflex responses to vagal afferent stimulation and nTS glutamate nanoinjection. Blunted responses occur via reduced presynaptic Ca2+ activation and attenuated NMDA receptor expression and function, leading to a reduction in nTS neuronal activation. These results provide insight into the control of autonomic and respiratory function, as well as the plasticity that can occur in response to nerve damage and cardiorespiratory disease.

Paraventricular nucleus projections to the nucleus tractus solitarii are essential for full expression of hypoxia-induced peripheral chemoreflex responses.

The hypothalamic paraventricular nucleus (PVN) is essential to peripheral chemoreflex neurocircuitry, but the specific efferent pathways utilized are not well defined. The PVN sends dense projections to the nucleus tractus solitarii (nTS), which exhibits neuronal activation following a hypoxic challenge. We hypothesized that nTS-projecting PVN (PVN-nTS) neurons contribute to hypoxia-induced nTS neuronal activation and cardiorespiratory responses. To selectively target PVN-nTS neurons, rats underwent bilateral nTS nanoinjection of retrogradely transported adeno-associated virus (AAV) driving Cre recombinase expression. We then nanoinjected into PVN AAVs driving Cre-dependent expression of Gq or Gi designer receptors exclusively activated by designer drugs (DREADDs) to test the degree that selective activation or inhibition, respectively, of the PVN-nTS pathway affects the hypoxic ventilatory response (HVR) of conscious rats. We used immunohistochemistry for Fos and extracellular recordings to examine how DREADD activation influences PVN-nTS neuronal activation by hypoxia. Pathway activation enhanced the HVR at moderate hypoxic intensities and increased PVN and nTS Fos immunoreactivity in normoxia and hypoxia. In contrast, PVN-nTS inhibition reduced both the HVR and PVN and nTS neuronal activation following hypoxia. To further confirm selective pathway effects on central cardiorespiratory output, rats underwent hypoxia before and after bilateral nTS nanoinjections of C21 to activate or inhibit PVN-nTS terminals. PVN terminal activation within the nTS enhanced tachycardic, sympathetic and phrenic (PhrNA) nerve activity responses to hypoxia whereas inhibition attenuated hypoxia-induced increases in nTS neuronal action potential discharge and PhrNA. The results demonstrate the PVN-nTS pathway enhances nTS neuronal activation and is necessary for full cardiorespiratory responses to hypoxia. KEY POINTS: The hypothalamic paraventricular nucleus (PVN) contributes to peripheral chemoreflex cardiorespiratory responses, but specific PVN efferent pathways are not known. The nucleus tractus solitarii (nTS) is the first integration site of the peripheral chemoreflex, and the nTS receives dense projections from the PVN. Selective GqDREADD activation of the PVN-nTS pathway was shown to enhance ventilatory responses to hypoxia and activation (Fos immunoreactivity (IR)) of nTS neurons in conscious rats, augmenting the sympathetic and phrenic nerve activity (SSNA and PhrNA) responses to hypoxia in anaesthetized rats. Selective GiDREADD inhibition of PVN-nTS neurons attenuates ventilatory responses, nTS neuronal Fos-IR, action potential discharge and PhrNA responses to hypoxia. These results demonstrate that a projection from the PVN to the nTS is critical for full chemoreflex responses to hypoxia.

Nucleus tractus solitarii is required for the development and maintenance of phrenic and sympathetic long-term facilitation after acute intermittent hypoxia.

Exposure to acute intermittent hypoxia (AIH) induces prolonged increases (long term facilitation, LTF) in phrenic and sympathetic nerve activity (PhrNA, SNA) under basal conditions, and enhanced respiratory and sympathetic responses to hypoxia. The mechanisms and neurocircuitry involved are not fully defined. We tested the hypothesis that the nucleus tractus solitarii (nTS) is vital to augmentation of hypoxic responses and the initiation and maintenance of elevated phrenic (p) and splanchnic sympathetic (s) LTF following AIH. nTS neuronal activity was inhibited by nanoinjection of the GABAA receptor agonist muscimol before AIH exposure or after development of AIH-induced LTF. AIH but not sustained hypoxia induced pLTF and sLTF with maintained respiratory modulation of SSNA. nTS muscimol before AIH increased baseline SSNA with minor effects on PhrNA. nTS inhibition also markedly blunted hypoxic PhrNA and SSNA responses, and prevented altered sympathorespiratory coupling during hypoxia. Inhibiting nTS neuronal activity before AIH exposure also prevented the development of pLTF during AIH and the elevated SSNA after muscimol did not increase further during or following AIH exposure. Furthermore, nTS neuronal inhibition after the development of AIH-induced LTF substantially reversed but did not eliminate the facilitation of PhrNA. Together these findings demonstrate that mechanisms within the nTS are critical for initiation of pLTF during AIH. Moreover, ongoing nTS neuronal activity is required for full expression of sustained elevations in PhrNA following exposure to AIH although other regions likely also are important. Together, the data indicate that AIH-induced alterations within the nTS contribute to both the development and maintenance of pLTF.

The effect of orexin on the hypoxic ventilatory response of female rats is greatest in the active phase during diestrus.

We recently showed that in male rats, orexin contributes to the hypoxic ventilatory response (HVR), with a stronger effect in the active phase. The effect of orexin on the HVR in females has not been investigated. As estrogen can inhibit orexin neurons, here we hypothesized that orexin neurons are activated by hypoxia and facilitate the HVR only in diestrus, when estrogen is low. We exposed female rats (n = 10) to near-isocapnic hypoxia ([Formula: see text] from 0.21 to 0.09) over ∼5 min, after vehicle and again after suvorexant (a dual OxR antagonist; 20 mg/kg ip), with ventilation measured using whole body plethysmography. Each rat was tested in proestrus or estrus (p/estrus), and again in diestrus, during both inactive and active phases. We also performed immunohistochemistry (IHC) to determine the proportion of orexin neurons activated by acute hypoxia during diestrus (n = 6) or proestrus/estrus (n = 6) in the active phase. In the inactive phase, the HVR was unaffected by OxR blockade, irrespective of estrus stage. In the active phase, the effect of OxR blockade depended on stage: the slope of the HVR was significantly reduced by OxR blockade only during diestrus. IHC revealed that hypoxia activated more orexin neurons during diestrus compared with p/estrus. We conclude that in females, orexin neurons are activated by hypoxia and contribute to the HVR only in diestrus when estrogen levels are low. Stage of the estrus cycle should be considered when examining the physiological function of orexin neurons in females.NEW & NOTEWORTHY We previously showed that orexin facilitates the hypoxic ventilatory response (HVR) of adult male rats during the active phase. Others have shown that estrogen inhibits orexin neurons. Here we show that orexin neurons are activated by hypoxia and facilitate the HVR of adult female rats during the active phase, but only in diestrus. These data suggest that orexin neurons facilitate the HVR in females when they are free from the inhibitory effects of estrogen.

Cardiovascular deconditioning increases GABA signaling in the nucleus tractus solitarii.

The nucleus tractus solitarii (nTS) is the major integrative brainstem region for autonomic modulation and processing of cardiovascular reflexes. GABA and glutamate are the main inhibitory and excitatory neurotransmitters, respectively, within this nucleus. Alterations in the GABA-glutamate regulation in the nTS are related to numerous cardiovascular comorbidities. Bedridden individuals and people exposed to microgravity exhibit dysautonomia and cardiovascular deconditioning that are mimicked in the hindlimb unloading (HU) rat model. We have previously shown in the nTS that HU increases glutamatergic neurotransmission yet decreases neuronal excitability. In this study, we investigated the effects of HU on nTS GABAergic neurotransmission. We hypothesized that HU potentiates GABA signaling via increased GABAergic release and postsynaptic GABA receptor expression. Following HU or control postural exposure, GABAergic neurotransmission was assessed using whole cell patch clamp whereas the magnitude of GABA release was evaluated via an intensity-based GABA sensing fluorescence reporter (iGABASnFR). In response to GABA interneuron stimulation, the evoked inhibitory postsynaptic current (nTS-IPSC) amplitude and area, as well as iGABASnFR fluorescence, were greater in HU than in control. HU also elevated the frequency but not the amplitude of spontaneous miniature IPSCs. Picoapplication of GABA produced similar postsynaptic current responses in nTS neurons of HU and control. Moreover, HU did not alter GABAA receptor α1 subunit expression, indicating minimal alterations in postsynaptic membrane receptor expression. These results indicate that HU increases GABAergic signaling in the nTS likely via augmented release of GABA from presynaptic terminals. Altogether, our data indicate GABA plasticity contributes to the autonomic and cardiovascular alterations following cardiovascular deconditioning (CVD).NEW & NOTEWORTHY Gravity influences distribution of blood volume and autonomic function. Microgravity and prolonged bed rest induce cardiovascular deconditioning (CVD). We used hindlimb unloading (HU), a rat analog for bed rest, to investigate CVD-induced neuroplasticity in the brainstem. Our data demonstrate that HU increases GABA modulation of nucleus tractus solitarii (nTS) neurons via presynaptic plasticity. Given the importance of nTS in integrating cardiovascular reflexes, this study provides new evidence on the central mechanisms behind CVD following HU.

Orexin facilitates the ventilatory and behavioral responses of rats to hypoxia.

Orexin neurons are sensitive to CO2 and contribute to cardiorespiratory homeostasis as well as sensorimotor control. Whether orexin facilitates respiratory and behavioral responses to acute hypoxia is unclear. We hypothesized that orexin neurons are activated by acute hypoxia and that orexin facilitates the hypoxic ventilatory response (HVR), as well as the arterial blood pressure (ABP) and behavioral (movement) responses to acute hypoxia. We further hypothesized that orexin has greater effects in the active phase of the rat circadian cycle, when orexin neurons have high activity. Using whole body plethysmography with EEG, EMG, and the dual-orexin receptor (OxR) antagonist suvorexant (20 mg/kg ip), we determined the effect of OxR blockade on the respiratory, ABP, and behavioral responses of adult rats to acute, graded hypoxia ([Formula: see text]= 0.15, 0.13, 0.11, and 0.09) and hyperoxic hypercapnia ([Formula: see text]= 0.05; [Formula: see text]= 0.95). OxR blockade had no effect on eupnea. OxR blockade significantly reduced the HVR in both inactive and active phases, with a stronger effect in the active phase. OxR blockade reduced the behavioral response to acute hypoxia in the active phase. The central component of the ventilatory and the ABP responses to hypercapnia were reduced by OxR blockade solely in the inactive phase. In the inactive phase, hypoxia activated ∼10% of orexin neurons in the perifornical hypothalamus. These data suggest that orexin neurons participate in the peripheral chemoreflex to facilitate the ventilatory and behavioral responses to acute hypoxia in rats, particularly in the active phase. Orexin also facilitates central chemoreflex responses to CO2 in the inactive phase.

Unilateral vagotomy alters astrocyte and microglial morphology in the nucleus tractus solitarii of the rat.

The nucleus tractus solitarii (nTS) is the initial site of integration of sensory information from the cardiorespiratory system and contributes to reflex responses to hypoxia. Afferent fibers of the bilateral vagus nerves carry input from the heart, lungs, and other organs to the nTS where it is processed and modulated. Vagal afferents and nTS neurons are integrally associated with astrocytes and microglia that contribute to neuronal activity and influence cardiorespiratory control. We hypothesized that vagotomy would alter glial morphology and cardiorespiratory responses to hypoxia. Unilateral vagotomy (or sham surgery) was performed in rats. Prior to and seven days after surgery, baseline and hypoxic cardiorespiratory responses were monitored in conscious and anesthetized animals. The brainstem was sectioned and caudal, mid-area postrema (mid-AP), and rostral sections of the nTS were prepared for immunohistochemistry. Vagotomy increased immunoreactivity (-IR) of astrocytic glial fibrillary acidic protein (GFAP), specifically at mid-AP in the nTS. Similar results were found in the dorsal motor nucleus of the vagus (DMX). Vagotomy did not alter nTS astrocyte number, yet increased astrocyte branching and altered morphology. In addition, vagotomy both increased nTS microglia number and produced morphologic changes indicative of activation. Cardiorespiratory baseline parameters and hypoxic responses remained largely unchanged, but vagotomized animals displayed fewer augmented breaths (sighs) in response to hypoxia. Altogether, vagotomy alters nTS glial morphology, indicative of functional changes in astrocytes and microglia that may affect cardiorespiratory function in health and disease.

Gamma-Aminobutyric Acid Transporters in the Nucleus Tractus Solitarii Regulate Inhibitory and Excitatory Synaptic Currents That Influence Cardiorespiratory Function.

The brainstem nucleus tractus solitarii (nTS) processes and modulates the afferent arc of critical peripheral cardiorespiratory reflexes. Sensory afferents release glutamate to initiate the central component of these reflexes, and glutamate concentration is critically controlled by its removal via astrocytic neurotransmitter transporters. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the nTS providing tonic and phasic modulation of neuronal activity. GABA is removed from the extracellular space through GABA transporters (GATs), however, the role of GATs in nTS synaptic transmission and their influence on cardiorespiratory function is unknown. We hypothesized that GATs tonically restrain nTS inhibitory signaling and given the considerable nTS GABA-glutamate cross-talk, modify excitatory signaling and thus cardiorespiratory function. Reverse transcription real-time polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry showed expression of GAT-1 and GAT-3 mRNA and protein within the rat nTS, with GAT-3 greater than GAT-1, and GAT-3 colocalizing with astrocyte S100B. Recordings in rat nTS slices demonstrated GAT-3 block decreased spontaneous inhibitory postsynaptic current (IPSC) frequency and reduced IPSC amplitude evoked from electrical stimulation of the medial nTS. Block of GAT-3 also increased spontaneous excitatory postsynaptic current (EPSC) frequency yet did not alter sensory afferent-evoked EPSC amplitude. Block of GAT-3 in the nTS of anesthetized rats increased mean arterial pressure, heart rate, sympathetic nerve activity, and minute phrenic nerve activity. These results demonstrate inhibitory and excitatory neurotransmission in the nTS is significantly modulated by endogenous GAT-3 to influence basal cardiorespiratory function.

Mechanisms Underlying Neuroplasticity in the Nucleus Tractus Solitarii Following Hindlimb Unloading in Rats.

Hindlimb unloading (HU) in rats induces cardiovascular deconditioning (CVD) analogous to that observed in individuals exposed to microgravity or bed rest. Among other physiological changes, HU rats exhibit autonomic imbalance and altered baroreflex function. Lack of change in visceral afferent activity that projects to the brainstem in HU rats suggests that neuronal plasticity within central nuclei processing cardiovascular afferents may be responsible for these changes in CVD and HU. The nucleus tractus solitarii (nTS) is a critical brainstem region for autonomic control and integration of cardiovascular reflexes. In this study, we used patch electrophysiology, live-cell calcium imaging and molecular methods to investigate the effects of HU on glutamatergic synaptic transmission and intrinsic properties of nTS neurons. HU increased the amplitude of monosynaptic excitatory postsynaptic currents and presynaptic calcium entry evoked by afferent tractus solitarii stimulus (TS-EPSC); spontaneous (s) EPSCs were unaffected. The addition of a NMDA receptor antagonist (AP5) reduced TS-EPSC amplitude and sEPSC frequency in HU but not control. Despite the increase in glutamatergic inputs, HU neurons were more hyperpolarized and exhibited intrinsic decreased excitability compared to controls. After block of ionotropic glutamatergic and GABAergic synaptic transmission (NBQX, AP5, Gabazine), HU neuronal membrane potential depolarized and neuronal excitability was comparable to controls. These data demonstrate that HU increases presynaptic release and TS-EPSC amplitude, which includes a NMDA receptor component. Furthermore, the decreased excitability and hyperpolarized membrane after HU are associated with enhanced GABAergic modulation. This functional neuroplasticity in the nTS may underly the CVD induced by HU.

Loss of excitatory amino acid transporter restraint following chronic intermittent hypoxia contributes to synaptic alterations in nucleus tractus solitarii.

Peripheral viscerosensory afferent signals are transmitted to the nucleus tractus solitarii (nTS) via release of glutamate. Following release, glutamate is removed from the extrasynaptic and synaptic cleft via excitatory amino acid transporters (EAATs), thus limiting glutamate receptor activation or over activation, and maintaining its working range. We have shown that EAAT block with the antagonist threo-ß-benzyloxyaspartic acid (TBOA) depolarized nTS neurons and increased spontaneous excitatory postsynaptic current (sEPSC) frequency yet reduced the amplitude of afferent (TS)-evoked EPSCs (TS-EPSCs). Interestingly, chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea (OSA), produces similar synaptic responses as EAAT block. We hypothesized EAAT expression or function are downregulated after CIH, and this reduction in glutamate removal contributes to the observed neurophysiological responses. To test this hypothesis, we used brain slice electrophysiology and imaging of glutamate release and TS-afferent Ca2+ to compare nTS properties of rats exposed to 10 days of normoxia (Norm; 21%O2) or CIH. Results show that EAAT blockade with (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]-amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) in Norm caused neuronal depolarization, generation of an inward current, and increased spontaneous synaptic activity. The latter augmentation was eliminated by inclusion of tetrodotoxin in the perfusate. TS stimulation during TFB-TBOA also elevated extracellular glutamate and decreased presynaptic Ca2+ and TS-EPSC amplitude. In CIH, the effects of EAAT block are eliminated or attenuated. CIH reduced EAAT expression in nTS, which may contribute to the attenuated function seen in this condition. Therefore, CIH reduces EAAT influence on synaptic and neuronal activity, which may lead to the physiological consequences seen in OSA and CIH.NEW & NOTEWORTHY Removal of excitatory amino acid transporter (EAAT) restraint increases spontaneous synaptic activity yet decreases afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. In the chronic intermittent hypoxia model of obstructive sleep apnea, this restraint is lost due to reduction in EAAT expression and function. Thus EAATs are important in controlling elevated glutamatergic signaling, and loss of such control results in maladaptive synaptic signaling.

Thrombin action on astrocytes in the hindbrain of the rat disrupts glycemic and respiratory control.

Severe trauma can produce a postinjury "metabolic self-destruction" characterized by catabolic metabolism and hyperglycemia. The severity of the hyperglycemia is highly correlated with posttrauma morbidity and mortality. Although no mechanism has been posited to connect severe trauma with a loss of autonomic control over metabolism, traumatic injury causes other failures of autonomic function, notably, gastric stasis and ulceration ("Cushing's ulcer"), which has been connected with the generation of thrombin. Our previous studies established that proteinase-activated receptors (PAR1; "thrombin receptors") located on astrocytes in the autonomically critical nucleus of the solitary tract (NST) can modulate gastric control circuit neurons to cause gastric stasis. Hindbrain astrocytes have also been implicated as important detectors of low glucose or glucose utilization. When activated, these astrocytes communicate with hindbrain catecholamine neurons that, in turn, trigger counterregulatory responses (CRR). There may be a convergence between the effects of thrombin to derange hindbrain gastrointestinal control and the hindbrain circuitry that initiates CRR to increase glycemia in reaction to critical hypoglycemia. Our results suggest that thrombin acts within the NST to increase glycemia through an astrocyte-dependent mechanism. Blockade of purinergic gliotransmission pathways interrupted the effect of thrombin to increase glycemia. Our studies also revealed that thrombin, acting in the NST, produced a rapid, dramatic, and potentially lethal suppression of respiratory rhythm that was also a function of purinergic gliotransmission. These results suggest that the critical connection between traumatic injury and a general collapse of autonomic regulation involves thrombin action on astrocytes.

Sustained Hypoxia Alters nTS Glutamatergic Signaling and Expression and Function of Excitatory Amino Acid Transporters.

Glutamate is the major excitatory neurotransmitter in the nucleus tractus solitarii (nTS) and mediates chemoreflex function during periods of low oxygen (i.e. hypoxia). We have previously shown that nTS excitatory amino acid transporters (EAATs), specifically EAAT-2, located on glia modulate neuronal activity, cardiorespiratory and chemoreflex function under normal conditions via its tonic uptake of extracellular glutamate. Chronic sustained hypoxia (SH) elevates nTS synaptic transmission and chemoreflex function. The goal of this study was to determine the extent to which glial EAAT-2 contributes to SH-induced nTS synaptic alterations. To do so, male Sprague-Dawley rats (4-7 weeks) were exposed to either 1, 3, or 7 days of SH (10% O2, 24 h/day) and compared to normoxic controls (21% O2, 24 h/day, i.e., 0 days SH). After which, the nTS was harvested for patch clamp electrophysiology, quantitative real-time PCR, immunohistochemistry and immunoblots. SH induced time- and parameter-dependent increases in excitatory postsynaptic currents (EPSCs). TS-evoked EPSC amplitude increased after 1D SH which returned at 3D and 7D SH. Spontaneous EPSC frequency increased only after 3D SH, which returned to normoxic levels at 7D SH. EPSC enhancement occurred primarily by presynaptic mechanisms. Inhibition of EAAT-2 with dihydrokainate (DHK, 300 µM) did not alter EPSCs following 1D SH but induced depolarizing inward currents (Ihold). After 3D SH, DHK decreased TS-EPSC amplitude yet its resulting Ihold was eliminated. EAAT-2 mRNA and protein increased after 3D and 7D SH, respectively. These data suggest that SH alters the expression and function of EAAT-2 which may have a neuroprotective effect.

Astrocytic glutamate transporters reduce the neuronal and physiological influence of metabotropic glutamate receptors in nucleus tractus solitarii.

Astrocytic excitatory amino acid transporters (EAATs) are critical to restraining synaptic and neuronal activity in the nucleus tractus solitarii (nTS). Relief of nTS EAAT restraint generates two opposing effects, an increase in neuronal excitability that reduces blood pressure and breathing and an attenuation in afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. Although the former is due, in part, to activation of ionotropic glutamate receptors, there remains a substantial contribution from another unidentified glutamate receptor. In addition, the mechanism(s) by which EAAT inhibition reduced TS-EPSC amplitude is unknown. Metabotropic glutamate receptors (mGluRs) differentially modulate nTS excitability. Activation of group I mGluRs on nTS neuron somas leads to depolarization, whereas group II/III mGluRs on sensory afferents decrease TS-EPSC amplitude. Thus we hypothesize that EAATs control postsynaptic excitability and TS-EPSC amplitude via restraint of mGluR activation. To test this hypothesis, we used in vivo recording, brain slice electrophysiology, and imaging of glutamate release and TS-afferent Ca2+. Results show that EAAT blockade in the nTS with (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) induced group I mGluR-mediated depressor, bradycardic, and apneic responses that were accompanied by neuronal depolarization, elevated discharge, and increased spontaneous synaptic activity. Conversely, upon TS stimulation TFB-TBOA elevated extracellular glutamate to decrease presynaptic Ca2+ and TS-EPSC amplitude via activation of group II/III mGluRs. Together, these data suggest an important role of EAATs in restraining mGluR activation and overall cardiorespiratory function.

The PVN enhances cardiorespiratory responses to acute hypoxia via input to the nTS.

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.

Hypoxia activates a neuropeptidergic pathway from the paraventricular nucleus of the hypothalamus to the nucleus tractus solitarii.

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% O2, n = 5) or hypoxia (10% O2, 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.

Glial EAAT2 regulation of extracellular nTS glutamate critically controls neuronal activity and cardiorespiratory reflexes.

KEY POINTS: Excitatory amino acid transporter 2 (EAAT2) is present on astrocytes in the nucleus tractus solitarii (nTS), an important nucleus in cardiorespiratory control. Its specific role in influencing nTS neuronal activity and thereby basal and reflex cardiorespiratory function is unknown. The specific role of nTS EAAT2 was determined via whole animal and brainstem slice patch clamp experiments. Astrocytic EAAT2 buffers basal glutamate activation of AMPA-type glutamate receptors and therefore decreases baseline excitability of nTS neurons. EAAT2 modulates cardiorespiratory control and tempers excitatory cardiorespiratory responses to activation of the peripheral chemoreflex. This study supports the concept that nTS astrocyte transporters influence sympathetic nervous system activity and cardiorespiratory reflex function in health and disease. ABSTRACT: Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, its influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2-specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK-induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex-mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function.

Acute hypoxia activates neuroendocrine, but not presympathetic, neurons in the paraventricular nucleus of the hypothalamus: differential role of nitric oxide.

Hypoxia results in decreased arterial Po2, 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% O2) 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.

Excitatory amino acid transporters tonically restrain nTS synaptic and neuronal activity to modulate cardiorespiratory function.

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-ß-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.