Acute kidney injury is highly prevalent and associated with high morbidity and mortality, and there are no approved drugs for its prevention and treatment. Vagus nerve stimulation (VNS) alleviates ...inflammatory diseases including kidney disease; however, neural circuits involved in VNS-induced tissue protection remain poorly understood. The vagus nerve, a heterogeneous group of neural fibers, innervates numerous organs. VNS broadly stimulates these fibers without specificity. We used optogenetics to selectively stimulate vagus efferent or afferent fibers. Anterograde efferent fiber stimulation or anterograde (centripetal) sensory afferent fiber stimulation both conferred kidney protection from ischemia-reperfusion injury. We identified the C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis as the downstream pathway of vagus afferent fiber stimulation. Our study provides a map of the neural circuits important for kidney protection induced by VNS, which is critical for the safe and effective clinical application of VNS for protection from acute kidney injury.
The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. ...An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed
hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker,
mRNA (
). Most (∼75%) RTN neurons express low-to-moderate levels of
and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, TASK-2 and Gpr4. These
-low RTN neurons also express varying levels of transcripts for
,
, and
, and receptors for substance P, orexin, serotonin, and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of
mRNA. These
-high RTN neurons do not express
after hypercapnia and have low-to-undetectable levels of
or
transcripts; they also express
, but are essentially devoid of
and
transcripts. In male rats,
is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum,
is a selective marker of the RTN in rodents;
-low neurons, the vast majority, are central respiratory chemoreceptors, whereas
-high neurons likely have other functions.
Central respiratory chemoreceptors regulate arterial PCO
by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed
hybridization, we show here that a single marker,
mRNA (
), identifies RTN neurons in rodents. We also suggest that >75% of these
neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors (
and
). The other RTN neurons express very high levels of
, but low levels of
, and do not respond to hypercapnia. Their function is unknown.
Current understanding of the contribution of C1 neurons to blood pressure (BP) regulation derives predominantly from experiments performed in anesthetized animals or reduced
preparations. Here, we ...use ArchaerhodopsinT3.0 (ArchT) loss-of-function optogenetics to explore BP regulation by C1 neurons in intact, unanesthetized rats. Using a lentivirus that expresses ArchT under the Phox2b-activated promoter PRSx8 (PRSx8-ArchT), ∼65% of transduced neurons were C1 (balance retrotrapezoid nucleus, RTN). Other rats received CaMKII-ArchT3.0 AAV2 (CaMKII-ArchT), which transduced C1 neurons and larger numbers of unidentified glutamatergic and GABAergic cells. Under anesthesia, ArchT photoactivation reduced sympathetic nerve activity and BP and silenced/strongly inhibited most (7/12) putative C1 neurons. In unanesthetized PRSx8-ArchT-treated rats breathing room air, bilateral ArchT photoactivation caused a very small BP reduction that was only slightly larger under hypercapnia (6% FiCO
), but was greatly enhanced during hypoxia (10 and 12% FiO
), after sino-aortic denervation, or during isoflurane anesthesia. The degree of hypotension correlated with percentage of ArchT-transduced C1 neurons. ArchT photoactivation produced similar BP changes in CaMKII-ArchT-treated rats. Photoactivation in PRSX8-ArchT rats reduced breathing frequency (
), whereas
increased in CaMKII-ArchT rats. We conclude that the BP drop elicited by ArchT activation resulted from C1 neuron inhibition and was unrelated to breathing changes. C1 neurons have low activity under normoxia, but their activation is important to BP stability during hypoxia or anesthesia and contributes greatly to the hypertension caused by baroreceptor deafferentation. Finally, C1 neurons are marginally activated by hypercapnia and the large breathing stimulation caused by this stimulus has very little impact on resting BP.
C1 neurons are glutamatergic/peptidergic/catecholaminergic neurons located in the medulla oblongata, which may operate as a switchboard for differential, behavior-appropriate activation of selected sympathetic efferents. Based largely on experimentation in anesthetized or reduced preparations, a rostrally located subset of C1 neurons may contribute to both BP stabilization and dysregulation (hypertension). Here, we used Archaerhodopsin-based loss-of-function optogenetics to explore the contribution of these neurons to BP in conscious rats. The results suggest that C1 neurons contribute little to resting BP under normoxia or hypercapnia, C1 neuron discharge is restrained continuously by arterial baroreceptors, and C1 neuron activation is critical to stabilize BP under hypoxia or anesthesia. This optogenetic approach could also be useful to explore the role of C1 neurons during specific behaviors or in hypertensive models.
Hemorrhage initially triggers a rise in sympathetic nerve activity (SNA) that maintains blood pressure (BP); however, SNA is suppressed following severe blood loss causing hypotension. We ...hypothesized that adrenergic C1 neurons in the rostral ventrolateral medulla (C1RVLM) drive the increase in SNA during compensated hemorrhage, and a reduction in C1RVLM contributes to hypotension during decompensated hemorrhage. Using fiber photometry, we demonstrate that C1RVLM activity increases during compensated hemorrhage and falls at the onset of decompensated hemorrhage. Using optogenetics combined with direct recordings of SNA, we show that C1RVLM activation mediates the rise in SNA and contributes to BP stability during compensated hemorrhage, whereas a suppression of C1RVLM activity is associated with cardiovascular collapse during decompensated hemorrhage. Notably, re-activating C1RVLM during decompensated hemorrhage restores BP to normal levels. In conclusion, C1 neurons are a nodal point for the sympathetic response to blood loss.
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•C1 neurons monitor blood pressure in freely behaving rats•The response of C1 neurons to hemorrhage is biphasic•Inhibition of C1 during compensated hemorrhage causes hypotension•Activation of C1 restores blood pressure during decompensated hemorrhage
Souza et al. show that adrenergic C1 neurons in the caudal brainstem are required for the compensatory response to hemorrhage. A reduction in C1 activity leads to hypotension preceding circulatory shock. This work shows that C1 neurons are a nodal point for the sympathetic response to blood loss.
In conscious mammals, hypoxia or hypercapnia stimulates breathing while theoretically exerting opposite effects on central respiratory chemoreceptors (CRCs). We tested this theory by examining how ...hypoxia and hypercapnia change the activity of the retrotrapezoid nucleus (RTN), a putative CRC and chemoreflex integrator. Archaerhodopsin-(Arch)-transduced RTN neurons were reversibly silenced by light in anesthetized rats. We bilaterally transduced RTN and nearby C1 neurons with Arch (PRSx8-ArchT-EYFP-LVV) and measured the cardiorespiratory consequences of Arch activation (10 s) in conscious rats during normoxia, hypoxia, or hyperoxia. RTN photoinhibition reduced breathing equally during non-REM sleep and quiet wake. Compared with normoxia, the breathing frequency reduction (Δf(R)) was larger in hyperoxia (65% FiO2), smaller in 15% FiO2, and absent in 12% FiO2. Tidal volume changes (ΔV(T)) followed the same trend. The effect of hypoxia on Δf(R) was not arousal-dependent but was reversed by reacidifying the blood (acetazolamide; 3% FiCO2). Δf(R) was highly correlated with arterial pH up to arterial pH (pHa) 7.5 with no frequency inhibition occurring above pHa 7.53. Blood pressure was minimally reduced suggesting that C1 neurons were very modestly inhibited. In conclusion, RTN neurons regulate eupneic breathing about equally during both sleep and wake. RTN neurons are the first putative CRCs demonstrably silenced by hypocapnic hypoxia in conscious mammals. RTN neurons are silent above pHa 7.5 and increasingly active below this value. During hyperoxia, RTN activation maintains breathing despite the inactivity of the carotid bodies. Finally, during hypocapnic hypoxia, carotid body stimulation increases breathing frequency via pathways that bypass RTN.
Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity
. The neural systems that regulate ...breathing are fragile early in development, and it is not clear how they adjust to support breathing at birth. Here we identify a neuropeptide system that becomes activated immediately after birth and supports breathing. Mice that lack PACAP selectively in neurons of the retrotrapezoid nucleus (RTN) displayed increased apnoeas and blunted CO
-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor PAC1 from the pre-Bötzinger complex-an RTN target region responsible for generating the respiratory rhythm-phenocopied the breathing deficits observed after RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the pre-Bötzinger complex. Notably, a postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apnoeas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.
Collectively, the retrotrapezoid nucleus (RTN) and adjacent C1 neurons regulate breathing, circulation and the state of vigilance, but previous methods to manipulate the activity of these neurons ...have been insufficiently selective to parse out their relative roles. We hypothesize that RTN and C1 neurons regulate distinct aspects of breathing (e.g., frequency, amplitude, active expiration, sighing) and differ in their ability to produce arousal from sleep. Here we use optogenetics and a combination of viral vectors in adult male and female
-Cre rats to transduce selectively RTN (Phox2b
) or C1 neurons (Phox2b
/
) with Channelrhodopsin-2. RTN photostimulation modestly increased the probability of arousal. RTN stimulation robustly increased breathing frequency and amplitude; it also triggered strong active expiration but not sighs. Consistent with these responses, RTN innervates the entire pontomedullary respiratory network, including expiratory premotor neurons in the caudal ventral respiratory group, but RTN has very limited projections to brainstem regions that regulate arousal (locus ceruleus, CGRP
parabrachial neurons). C1 neuron stimulation produced robust arousals and similar increases in breathing frequency and amplitude compared with RTN stimulation, but sighs were elicited and active expiration was absent. Unlike RTN, C1 neurons innervate the locus ceruleus, CGRP
processes within the parabrachial complex, and lack projections to caudal ventral respiratory group. In sum, stimulating C1 or RTN activates breathing robustly, but only RTN neuron stimulation produces active expiration, consistent with their role as central respiratory chemoreceptors. Conversely, C1 stimulation strongly stimulates ascending arousal systems and sighs, consistent with their postulated role in acute stress responses.
The C1 neurons and the retrotrapezoid nucleus (RTN) reside in the rostral ventrolateral medulla. Both regulate breathing and the cardiovascular system but in ways that are unclear because of technical limitations (anesthesia, nonselective neuronal actuators). Using optogenetics in unanesthetized rats, we found that selective stimulation of either RTN or C1 neurons activates breathing. However, only RTN triggers active expiration, presumably because RTN, unlike C1, has direct excitatory projections to abdominal premotor neurons. The arousal potential of the C1 neurons is far greater than that of the RTN, however, consistent with C1's projections to brainstem wake-promoting structures. In short, C1 neurons orchestrate cardiorespiratory and arousal responses to somatic stresses, whereas RTN selectively controls lung ventilation and arterial Pco
stability.
The interoceptive homeostatic mechanism that controls breathing, blood gases and acid‐base balance in response to changes in CO2/H+ is exquisitely sensitive, with convergent roles proposed for ...chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na+‐HCO3− cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO2‐induced local extracellular acidification or purinergic signalling). We tested these NBCe1‐centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP‐Cre;Slc4a4fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1‐mediated current. Despite disrupted NBCe1 function in RTN‐adjacent astrocytes from these conditional knockout mice, CO2‐induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO2‐stimulated breathing, were indistinguishable from NBCe1‐intact littermates; hypoxia‐stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen‐treated Aldh1l1‐Cre/ERT2;Slc4a4fl/fl mice. Again, there was no difference in effects of CO2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1‐deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1‐independent mechanisms.
Key points
The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO2/H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing.
We used two different Cre mouse lines for cell‐specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis.
In both mouse lines, Slc4a4 was depleted from RTN‐associated astrocytes but CO2‐induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact.
Likewise, respiratory chemoreflexes evoked by changes in CO2 or O2 were unaffected by loss of astrocytic Slc4a4.
These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.
figure legend Astrocytic NBCe1 expression is not required for CO2‐stimuated breathing in mice. Mice bearing ‘floxed’ alleles for Slc4a4, the gene that encodes the NBCe1 electrogenic Na+‐HCO3‐ co‐transporter, were crossed with two different lines of mice that express Cre recombinase preferentially in astrocytes. After conditional knockout of Slc4a4, NBCe1‐like currents were eliminated from astrocytes, but CO2‐stimulated breathing was unaffected. Thus, contrary to a prominent current hypothesis, astrocytic NBCe1 does not appear to play a role in CO2‐stimulated breathing, and alternative mechanisms should be sought to explain proposed astrocytic contributions to this respiratory chemoreflex.
•In rats, arcuate nucleus Angiotensin II (AngII) and leptin are sympathoexcitatory.•Arcuate blockade of AngII type 1 receptors (AT1aR) prevents leptin-induced effects.•In rats, arcuate AT1aR neurons ...rarely co-express leptin receptors.•Therefore the interdependence of arcuate leptin and AT1aR must occur via a local neuronal network.
The action of leptin in brain to increase sympathetic nerve activity (SNA) and blood pressure depends upon functional Angiotensin II (AngII) type 1a receptors (AT1aR); however, the sites and mechanism of interaction are unknown. Here we identify one site, the hypothalamic arcuate nucleus (ArcN), since prior local blockade of AT1aR in the ArcN with losartan or candesartan in anesthetized male rats essentially eliminated the sympathoexcitatory and pressor responses to ArcN leptin nanoinjections. Unlike mice, in male and female rats, AT1aR and LepR rarely co-localized, suggesting that this interdependence occurs indirectly, via a local interneuron or network of neurons. ArcN leptin increases SNA by activating pro-opiomelanocortin (POMC) inputs to the PVN, but this activation requires simultaneous suppression of tonic PVN Neuropeptide Y (NPY) sympathoinhibition. Because AngII-AT1aR inhibits ArcN NPY neurons, we propose that loss of AT1aR suppression of NPY blocks leptin-induced increases in SNA; in other words, ArcN-AngII-AT1aR is a gatekeeper for leptin-induced sympathoexcitation. With obesity, both leptin and AngII increase; therefore, the increased AT1aR activation could open the gate, allowing leptin (and insulin) to drive sympathoexcitation unabated, leading to hypertension.
Key points
The retrotrapezoid nucleus (RTN) drives breathing proportionally to brain PCO2 but its role during various states of vigilance needs clarification.
Under normoxia, RTN lesions increased ...the arterial PCO2 set‐point, lowered the PO2 set‐point and reduced alveolar ventilation relative to CO2 production. Tidal volume was reduced and breathing frequency increased to a comparable degree during wake, slow‐wave sleep and REM sleep. RTN lesions did not produce apnoeas or disordered breathing during sleep.
RTN lesions in rats virtually eliminated the central respiratory chemoreflex (CRC) while preserving the cardiorespiratory responses to hypoxia; the relationship between CRC and number of surviving RTN Nmb neurons was an inverse exponential.
The CRC does not function without the RTN. In the quasi‐complete absence of the RTN and CRC, alveolar ventilation is reduced despite an increased drive to breathe from the carotid bodies.
The retrotrapezoid nucleus (RTN) is one of several CNS nuclei that contribute, in various capacities (e.g. CO2 detection, neuronal modulation) to the central respiratory chemoreflex (CRC). Here we test how important the RTN is to PCO2 homeostasis and breathing during sleep or wake. RTN Nmb‐positive neurons were killed with targeted microinjections of substance P–saporin conjugate in adult rats. Under normoxia, rats with large RTN lesions (92 ± 4% cell loss) had normal blood pressure and arterial pH but were hypoxic (−8 mmHg PaO2) and hypercapnic (+10 mmHg ). In resting conditions, minute volume (VE) was normal but breathing frequency (fR) was elevated and tidal volume (VT) reduced. Resting O2 consumption and CO2 production were normal. The hypercapnic ventilatory reflex in 65% FiO2 had an inverse exponential relationship with the number of surviving RTN neurons and was decreased by up to 92%. The hypoxic ventilatory reflex (HVR; FiO2 21–10%) persisted after RTN lesions, hypoxia‐induced sighing was normal and hypoxia‐induced hypotension was reduced. In rats with RTN lesions, breathing was lowest during slow‐wave sleep, especially under hyperoxia, but apnoeas and sleep‐disordered breathing were not observed. In conclusion, near complete RTN destruction in rats virtually eliminates the CRC but the HVR persists and sighing and the state dependence of breathing are unchanged. Under normoxia, RTN lesions cause no change in VE but alveolar ventilation is reduced by at least 21%, probably because of increased physiological dead volume. RTN lesions do not cause sleep apnoea during slow‐wave sleep, even under hyperoxia.
Key points
The retrotrapezoid nucleus (RTN) drives breathing proportionally to brain PCO2 but its role during various states of vigilance needs clarification.
Under normoxia, RTN lesions increased the arterial PCO2 set‐point, lowered the PO2 set‐point and reduced alveolar ventilation relative to CO2 production. Tidal volume was reduced and breathing frequency increased to a comparable degree during wake, slow‐wave sleep and REM sleep. RTN lesions did not produce apnoeas or disordered breathing during sleep.
RTN lesions in rats virtually eliminated the central respiratory chemoreflex (CRC) while preserving the cardiorespiratory responses to hypoxia; the relationship between CRC and number of surviving RTN Nmb neurons was an inverse exponential.
The CRC does not function without the RTN. In the quasi‐complete absence of the RTN and CRC, alveolar ventilation is reduced despite an increased drive to breathe from the carotid bodies.
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