The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO
/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
-HCO
cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO
-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;Slc4a4
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, CO
-induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO
-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;Slc4a4
mice. Again, there was no difference in effects of CO
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 CO
/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 CO
-induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO
or O
were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.
Background
Adrenergic neurons in the rostral ventrolateral medulla (RVLM), called C1 neurons, regulate vasomotor sympathetic nerve activity (SNA) and blood pressure (BP), and are thought to ...contribute to BP homeostasis during hemorrhage. In unanesthetized rats, hemorrhage has two sequential phases: 1) compensated, when SNA is markedly elevated and BP is maintained at nearly normal levels and 2) decompensated, when SNA is suppressed, and BP falls dramatically. We hypothesize that dynamic changes in C1 neuron activity determines the level of SNA and BP during hemorrhage.
Aim
To measure C1 neuron activity during hypotensive hemorrhage in non‐anesthetized freely behaving rats.
Methods
The real‐time activity of C1 neurons was monitored using a genetically encoded calcium indicator, jGCaMP7s, with in vivo fiber photometry in non‐anesthetized rats. A virus carrying a Cre‐dependent GCaMP7 transgene (pGP‐AAV1‐syn‐FLEX‐jGCaMP7s‐WPRE) was injected in the RVLM of adult female and male Th‐Cre rats and an optic fiber was implanted at the site of injection. Four weeks later, rats were instrumented for BP measurements and vascular access for blood withdrawal. The next day, the activity of C1 neurons was measured while the rats were subjected to a controlled hypotensive hemorrhage (18 ml/kg of blood withdrawal over 20 minutes, rate 0.3‐0.5 ml/min). Isosbestic‐corrected fluorescence (DF/F0) is expressed as a Z‐score based on a baseline recording period expressed as mean ± SD. Tests include Student's t‐test or repeated measures one‐way ANOVA. Differences were considered significant at p<0.05.
Results
Four weeks after the injection, GCaMP7 was expressed selectively in C1 neurons (76 ± 12 % of GCaMP7‐positive neurons expressed tyrosine hydroxylase and PNMT). Transient hypotension induced by intravenous sodium nitroprusside (SNP) activated C1 neurons (from ‐0.3 ± 0.6 to 11.3 ± 4 A.U., p=0.0002, n=8), whereas elevations in BP induced by intravenous phenylephrine (PE) did not consistently reduce C1 neuron activity (from 0.1 ± 0.3 to ‐0.9 ± 2.3 A.U., p=0.343, n=7). During hemorrhage, the activity of C1 neurons increases during the compensated phase (when BP is stable despite the blood loss) in relation to the pre‐hemorrhage period (from 0.17 ± 0.8 to 5.2 ± 2.1 A.U., p=0.0006, n=8). At the onset of the decompensated phase, the activity of C1 neurons drops dramatically in relation to the compensated phase (from 5.2 ± 2.1 to 2.9 ± 2.8 A.U., p=0.0017, n=8).
Conclusions
Our results indicate that C1 neurons have low resting activity and are very sensitive to hypotension in quite awake freely behaving rats. And the activation of C1 neurons is critical for BP stability during compensated hemorrhage and their inhibition contributes to the reduction in SNA and hypotension during decompensated hemorrhage.
Arousal in response to asphyxia is a life‐saving reflex that helps restore normal breathing and blood gas homeostasis. The carotid bodies (CB) are essential to hypoxia‐induced arousal whereas CNS ...serotonergic neurons and the lateral parabrachial nucleus contribute to CO2‐induced arousal. Here we wished to test whether the retrotrapezoid nucleus (RTN) is also implicated in CO2‐induced arousal. Our hypothesis was that RTN contributes to arousal elicited by hypercapnia but not hypoxia. The rationale is as follows. RTN is an important pluricellular CO2 detector that mediates the bulk of the hypercapnic ventilatory reflex (HCVR). RTN is likely absent since birth in central congenital hypoventilation syndrome, CCHS, a developmental disease in which the HCVR and asphyxia‐induced arousal are both greatly reduced. To test our hypothesis, we studied four groups of adult Sprague‐Dawley rats. In one group, we nearly completely destroyed RTN (“RTN lesion” group; n=8) with microinjections of saporin conjugated with substance‐P (2.4 ng/injection). The cognate control group (“RTN control”; n=7) received saline. The CBs were ablated in a third rat cohort (“CBx rats”; n=7). The fourth group (“CB control rats”; n=7) underwent sham surgery. Four weeks later, rats were placed in a plethysmograph chamber where breathing frequency and amplitude, EEG and EMG were recorded. Using mass flow controllers, the gas mixture perfusing the chamber was intermittently changed for exactly 1 min from normoxia (21% O2/balance N2) back to normoxia (control for flow interruption), from normoxia to hypercapnia (15% CO2/21% O2/balance N2), or from normoxia to hypoxia (10% O2, balance N2). Sleep survival curves representing the cumulative probability for SWS to persist after onset of the gas change were obtained. Two‐way ANOVA for repeated measures was used for statistical analysis of these curves. Sleep architecture was assessed by measuring the proportion of time the rats spent in SWS, REM sleep or quiet waking and the number of sleep stage transitions. Based on these measurements, sleep was unaffected by CBx or RTN lesion. However, the probability of SWS to persist 20 sec after the beginning of stimulus was significantly higher in RTN lesion rats compared to control (73.6 ± 14% vs. 54.8 ± 18 % at 20 sec from the beginning of stimulus). The corresponding figures at the 30 s time‐point were 57.8 ± 12 vs. 9.6 ± 13% and, at 40 sec, 40.1 ± 11 vs. 0 %. By contrast, RTN lesion had no detectable effect on hypoxia‐induced arousal. The arousal deficits elicited by CBx were different. CBx‐rats exposed to hypoxia had a significantly probability of SWS to persist compared to CB Control rats (89.9 ± 9 vs. 67.1 ± 9 % at 20 sec; 81.5 ± 15 vs. 56.4 ± 14 % at 30 sec, and 70.8 ± 16 vs. 39.9 ± 11 at 40 sec after the beginning of stimulus). In summary, we confirm the importance of the CBs to hypoxia‐induced arousal and demonstrate that arousal to hypercapnia is selectively reduced after RTN lesion. Neither CBx nor RTN lesions eliminated the arousal elicited by hypoxia or hypercapnia, however. Additional O2 and CO2 sensors (peripheral or central) therefore likely contribute to asphyxia‐induced arousal.
Support or Funding Information
National Institutes of Health (HL074011 and HL28785 to P.G.G.)
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Tonic sympathetic nerve activity (SNA) is necessary for the maintenance of arterial pressure (AP) under anesthesia, and this tone is dependent on supraspinal input. Over the last two centuries, ...numerous studies have collectively identified the rostral ventrolateral medulla (RVLM) as a critical region for neural control of AP and SNA in anesthetized animals, however the relevance of these results to conscious animals and the nature of the RVLM neurons responsible remain conjectural. The RVLM has direct projections onto sympathetic preganglionic neurons, but the evidence that this is the critical pathway for AP regulation and SNA in conscious animals is not definitive—and even if this is the principal BP regulatory pathway of the RVLM, the bulbospinal neurons that produce these effects are still debated.
Historically, the most prominent pressor candidate within the RVLM has been the rostral portion of the catecholaminergic C1 group. Rostral C1 neurons are glutamatergic, occupy a ventral region of the medulla that is coextensive with the vasomotor RVLM region, are barosensitive, directly innervate sympathetic preganglionic neurons, and activate sympathetic nerve discharge upon stimulation. Despite these data, optogenetic stimulation of C1 in conscious and anesthetized rodents has thus far failed to achieve the magnitude of increases in arterial pressure predicted by pharmacological stimulation of the RVLM.
Here, we show that stimulation of a non‐catecholaminergic population within the RVLM is sufficient to increase AP in conscious mice, even after C1 lesion. We then targeted bulbospinal neurons using a retrograde virus and demonstrated that optogenetic activation of only bulbospinal RVLM neurons increases AP in conscious mice, and in anesthetized mice, stimulation increases AP and renal sympathetic nerve activity. Unlike optogenetic stimulation of C1 neurons, stimulation of non‐catecholaminergic bulbospinal RVLM causes robust and sustained increases in AP in both conscious and anesthetized states, and sustained increases in SNA under anesthesia. This data suggests non‐catecholaminergic bulbospinal neurons are powerful activators of AP and SNA and may play an important role along with C1 in arterial pressure maintenance.
Support or Funding Information
American Heart Association Predoctoral Fellowship; University of Virginia Wagner Fellowship
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
RTN senses PCO2 and regulates breathing in a CO2‐dependent manner but the full extent of RTN's contribution to the CRC is not known. Lesions produced by microinjecting a substance‐P analog conjugated ...with saporin (SSP‐SAP) elicit small and/or transient CRC reductions in rats. Interpretative limitations are many: development of compensatory mechanisms, especially of carotid body origin, difficulty to identify the critical neurons and to lesion them sufficiently. As shown recently, neuromedin B (Nmb) seems to be a selective marker of the Phox2b+/Vglut2+ RTN neurons that contribute to the CRC. In the present study, we microinjected saline or SSPSAP (3 injections per side; ng/injection: 0.6, 1.2 or 2.4 ng) to destroy RTN in ~250 g Sprague‐Dawley male rats. Physiological experiments were conducted three weeks later in unanesthetized animals, following which we counted the surviving Nmb+ neurons using fluorescent ISH (FISH) in a 1/6 series of sections. SSP‐lesioned rats were regrouped post hoc into two clusters according to the number of surviving RTN Nmb+ neurons: mild lesion group (ML rats; N= 13; > 55 Nmb+ cells) and large lesion group (LL rats; N=11; 4–55 Nmb+ cells remaining). Control rats (C rats; N= 11) had 324 ± 48 Nmb+ neurons (these and all subsequent values represent mean ± SD). Under normoxia, arterial pH was virtually the same in all groups (LL rats: 7.46 ± 0.03; ML: 7.48 ± 0.01; C: 7.49 ± 0.01). PaO2 was lower in LL rats than in C and ML animals (71 ± 7 vs. 80 ± 6 vs. 78 ± 6 mmHg) but PaCO2 was higher (49 ± 7 vs. 38 ± 3 vs. 39 ± 5 mmHg). BP measured via arterial catheter was the same in all groups. Under normoxia, LL rats had a reduced VT relative to C and ML rats during quiet waking (0.39 ± 0.1 vs. 0.49 ± 0.07 vs. 0.54 ± 0.1 mL/100g), SW sleep (0.32 ± 0.08 vs.0.45 ± 0.06 vs. 0.50 ± 0.1 mL/100g) and REM sleep (0.25 ± 0.06 vs. 0.35±0.05 vs. 0.36 ± 0.1 mL/100g). Short‐term hyperoxia (1 min of 65% FiO2, balance N2) caused a larger decrease in VE in LL and in ML rats compared to C (ΔVE: −11 ± 3 vs. −13 ± 6 vs. −8 ± 3 mL/100g/min). Of note, the hypoventilation still present after 20 min of continuous hyperoxia and was larger in LL than in ML and C rats (ΔVE: −9 ± 6 vs. −5 ± 10 vs. 0.9 ± 4 mL/100g/min). When exposed to 6 % FiCO2 in 65% FiO2 LL rats had a greatly reduced breathing stimulation (hypercapnic ventilatory reflex, HCVR) compared to C and ML rats (ΔVE: 17.8 ± 9 vs. 63.6 ± 12 vs. 49.5 ± 21 mL/100g/min). The HCVR to 9 % FiCO2 in 65% FiO2 was reduced in similar proportion (ΔVE: 25.6 ± 9 vs. 85.9 ± 14 vs. 76.3 ± 27 mL/100g/min). Hypoxia (10% FiO2, balance N2) activated VE similarly in all three groups (ΔVE: 34.1 ± 6 vs. 27.2 ± 8 vs. 28.2 ± 9 in mL/100g/min for LL, C and ML rats respectively) although ΔVT was enhanced and ΔFR reduced in LL rats. In conclusion, >80% RTN Nmb+ neurons need to be destroyed to produce a notable reduction of the HCVR. Large RTN lesions reduce the CRC by ≥ 71% without decreasing the hypoxic ventilatory reflex. Large RTN lesions raise the arterial PCO2 homeostatic set‐point by ≥10 mmHg and lower PaO2 by ~9 mmHg but have little influence on long‐term arterial pH. Finally, the loss of breathing stimulation contributed by RTN is largely compensated by a permanent state of hypoxia and increased carotid body activity.
Support or Funding Information
National Institutes of Health (Grants RO1 HL074011 and RO1 HL 028785 to P.G.G.)
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
A long-standing theory posits that central chemoreception, the CNS mechanism for CO(2) detection and regulation of breathing, involves neurons located at the ventral surface of the medulla oblongata ...(VMS). Using in vivo and in vitro electrophysiological recordings, we identify VMS neurons within the rat retrotrapezoid nucleus (RTN) that have characteristics befitting these elusive chemoreceptors. These glutamatergic neurons are vigorously activated by CO(2) in vivo, whereas serotonergic neurons are not. Their CO(2) sensitivity is unaffected by pharmacological blockade of the respiratory pattern generator and persists without carotid body input. RTN CO(2)-sensitive neurons have extensive dendrites along the VMS and they innervate key pontomedullary respiratory centers. In brainstem slices, a subset of RTN neurons with markedly similar morphology is robustly activated by acidification and CO(2). Their pH sensitivity is intrinsic and involves a background K(+) current. In short, the CO(2)-sensitive neurons of the RTN are good candidates for the long sought-after VMS chemoreceptors.
The rat retrotrapezoid nucleus (RTN) contains neurons described as central chemoreceptors in the adult and respiratory rhythm-generating pacemakers in neonates parafacial respiratory group (pfRG). ...Here we test the hypothesis that both RTN and pfRG neurons are intrinsically chemosensitive and tonically firing neurons whose respiratory rhythmicity is caused by a synaptic feedback from the central respiratory pattern generator (CPG). In halothane-anesthetized adults, RTN neurons were silent below 4.5% end-expiratory (e-exp) CO2. Their activity increased linearly (3.2 Hz/1% CO2) up to 6.5% (CPG threshold) and then more slowly to peak approximately 10 Hz at 10% CO2. Respiratory modulation of RTN neurons was absent below CPG threshold, gradually stronger beyond, and, like pfRG neurons, typically (42%) characterized by twin periods of reduced activity near phrenic inspiration. After CPG inactivation with kynurenate (KYN), RTN neurons discharged linearly as a function of e-exp CO2 (slope, +1.7 Hz/1% CO2) and arterial pH (threshold, 7.48; slope, 39 Hz/pH unit). In coronal brain slices (postnatal days 7-12), RTN chemosensitive neurons were silent at pH 7.55. Their activity increased linearly with acidification up to pH 7.2 (17 Hz/pH unit at 35 degrees C) and was always tonic. In conclusion, consistent with their postulated central chemoreceptor role, RTN/pfRG neurons encode pH linearly and discharge tonically when disconnected from the rest of the respiratory centers in vivo (KYN treatment) and in vitro. In vivo, RTN neurons receive respiratory synchronous inhibitory inputs that may serve as feedback and impart these neurons with their characteristic respiratory modulation.
Central congenital hypoventilation syndrome is caused by mutations of the gene that encodes the transcription factor Phox2b. The syndrome is characterized by a severe form of sleep apnea attributed ...to greatly compromised central and peripheral chemoreflexes. In this study, we analyze whether Phox2b expression in the brainstem respiratory network is preferentially associated with neurons involved in chemosensory integration in rats. At the very rostral end of the ventral respiratory column (VRC), Phox2b was present in many VGlut2 (vesicular glutamate transporter 2) mRNA-containing neurons. These neurons were functionally identified as the respiratory chemoreceptors of the retrotrapezoid nucleus (RTN). More caudally in the VRC, many fewer neurons expressed Phox2b. These cells were not part of the central respiratory pattern generator (CPG), because they were typically cholinergic visceral motor neurons or catecholaminergic neurons (presumed C1 neurons). Phox2b was not detected in serotonergic neurons, in the A5, A6, and A7 noradrenergic cell groups nor within the main cardiorespiratory centers of the dorsolateral pons. Phox2b was expressed by many solitary tract nucleus (NTS) neurons including those that relay peripheral chemoreceptor information to the RTN. These and previous observations by others suggest that Phox2b is expressed by an uninterrupted chain of neurons involved in the integration of peripheral and central chemoreception (carotid bodies, chemoreceptor afferents, chemoresponsive NTS neurons projecting to VRC, RTN chemoreceptors). The presence of Phox2b in this circuit and its apparent absence from the respiratory CPG could explain why Phox2b mutations disrupt breathing automaticity during sleep without causing major impairment of respiration during waking.
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