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, 37 (17), 4565-4583

Blood Pressure Regulation by the Rostral Ventrolateral Medulla in Conscious Rats: Effects of Hypoxia, Hypercapnia, Baroreceptor Denervation, and Anesthesia

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Blood Pressure Regulation by the Rostral Ventrolateral Medulla in Conscious Rats: Effects of Hypoxia, Hypercapnia, Baroreceptor Denervation, and Anesthesia

Ian C Wenker et al. J Neurosci.

Abstract

Current understanding of the contribution of C1 neurons to blood pressure (BP) regulation derives predominantly from experiments performed in anesthetized animals or reduced ex vivo 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% FiCO2), but was greatly enhanced during hypoxia (10 and 12% FiO2), 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 (FR), whereas FR 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.SIGNIFICANCE STATEMENT 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.

Keywords: RVLM; baroreceptor; blood pressure; chemoreflex; optogenetics; sympathetic.

Figures

Figure 1.
Figure 1.
Phenotype and location of ArchT-transduced neurons. A, Example of ArchT-eYFP-expressing C1 neurons in a transverse section through the medulla oblongata of a rat injected with PRSx8-ArchT (level ∼11.9 mm caudal to bregma). A, TH immunoreactivity (magenta), ArchT-eYFP immunoreactivity (green), and overlay. Arrows indicate ArchT-transduced C1 neurons and arrowheads point to C1 neurons lacking ArchT. Scale bar, 50 μm. B, Example of ArchT-eYFP-expressing C1 neurons in a rat injected with CaMKII-ArchT. Arrows and arrowheads indicate ArchT-transduced C1 neurons and non-C1 neurons, respectively (medullary level, immunohistochemistry, color-coding, and scale bar same as in A). C, Rostrocaudal distribution of total TH+, TH+/eYFP+, and eYFP only cells in PRSx8-ArchT-injected rats (n = 7 rats, neurons/30 μm section counted on both sides; every sixth section counted; eYFP only cells were counted at three rostrocaudal levels). D, Cell counts of the same 3 classes of neurons in CaMKII-ArchT-injected rats (n = 7, neurons/30 μm section counted on both sides; every sixth section counted; eYFP only cells were counted at three rostrocaudal levels). E, RVLM neurons transduced with CaMKII-ArchT send dense projections selectively to the intermediolateral (IML) cell column of the spinal cord (transverse section). Scale bar, 200 μm. F, Simultaneous detection of ArchT-eYFP-ir (green), VGlut2 mRNA (magenta), and GAD1 mRNA (blue) in the RVLM of a rat injected with CaMKII-ArchT vector. Glutamatergic neurons (arrows) and GABAergic neurons (arrowheads) were both transduced. Scale bar, 50 μm.
Figure 2.
Figure 2.
Effect of ArchT photoactivation on the discharge of RVLM neurons. A, Single RVLM-barosensitive unit recorded in an α-chloralose/urethane anesthetized rat previously injected unilaterally with PRSx8-ArchT.The unit was 100% inhibited by raising BP with an intravenous injection of phenylephrine (PE, arrow) and silenced by 532 nm laser light (gray bar; 5 mW; 10 s). From top to bottom, End-expiratory CO2 (eeCO2), arterial BP, RVLM unit, and unit discharge rate. B, Identical experiment in a rat injected with CaMKII-ArchT. This RVLM-barosensitive neuron was also silenced by laser light (gray bar). In both cases, light-induced inhibition was followed by a brief rebound (arrowhead). C, Effect of light in seven barosensitive RVLM units recorded in animals injected with PRSx8-ArchT. D, Effect of light in five barosensitive RVLM units recorded in animals injected with CaMKII-ArchT. E, Distribution histogram representing the light-evoked change in unit activity of all RVLM neurons recorded: number of neurons are plotted versus the percentage change in unit activity binned in 10% increments from −100% (silenced units) to +100%.
Figure 3.
Figure 3.
Proton pump activation in RVLM suppresses SNA in anesthetized rats. A, Hypotension and inhibition of splanchnic SNA elicited by light (532 nm, 7 mW, applied for 10 s bilaterally at gray bars) in an α-chloralose/urethane anesthetized rat previously injected with CaMKII-ArchT. Traces from top to bottom, Arterial BP, HR, SNA (raw trace), and integrated SNA (Int SNA, 2 s time constant). SNA inhibition was modest at resting BP and enhanced when SNA was elevated by intravenous administration of sodium nitropusside (SNP, arrow). Arrowheads indicate SNA rebound immediately after the light was turned off. B, Group data (n = 6) depicting Int SNA before and during RVLM illumination under control conditions and shortly after intravenous injection of SNP. *p < 0.05; **p < 0.01.
Figure 4.
Figure 4.
Hypotension elicited by RVLM neuron inhibition is much larger in isoflurane-anesthetized rats than in conscious rats. A, Example recordings of BP, HR, and respiratory flow (Flow) from a rat previously injected with PRSx8-ArchT (A) and CaMKII-ArchT (B). Bilateral inhibition of RVLM neurons (gray bars) by ArchT had very little effect on BP in the absence of anesthetic (conscious state), but produced profound hypotension under ∼2% isoflurane anesthesia. In both rats, ArchT photoinhibition resulted in mild tachycardia in conscious rats and mild bradycardia under isoflurane. Respiratory responses were quite different between PRSx8-ArchT and CaMKII-ArchT rats. The former experienced decreased FR and VT in both conditions, whereas the latter increased FR in the conscious state. Arrowheads indicate rebound in BP after cessation of ArchT photoinhibition. CF, Data for all experimental animals (n = 7 for PRSx8-ArchT and n = 9 for CaMKII-ArchT) for MAP (C), HR (D), FR (E), and VT (F). Filled circles: 10 s before before bilateral ArchT photoactivation; open circles: during bilateral inhibition with ArchT. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 5.
Figure 5.
Effect of ArchT photoactivation on cardiorespiratory variables in conscious or anesthetized rats: comparison between animals injected with CaMKII-ArchT vs PRSx8-ArchT. AD, Average changes induced by proton pump activation (n = 7 for PRSx8-ArchT and n = 9 for CaMKII-ArchT). MAP (A), HR (B), FR (C), and VT (D) for rats injected with PRSx8-ArchT (black bars) and CaMKII-ArchT (gray bars).
Figure 6.
Figure 6.
Effect of ArchT photoactivation on MAP with varying levels of viral transduction in C1 neurons. A, Correlation between percentage C1 neurons transduced with PRSx8-ArchT and hypotension elicited by ArchT photoactivation (n = 20 rats, 7 high-responders and 13 other rats tested with low responses; C1 neurons counted between transverse planes 11.6 and 12.1 mm caudal to bregma; black triangles, unaesthetized rats; open circles: same rats during isoflurane anesthesia). B, Lack of correlation between percentage C1 neurons transduced with CaMKII-ArchT and hypotension elicited by ArchT photoactivation (n = 12 rats, 9 high-responders and 3 other rats tested with low responses). *, **, *** and **** indicate *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 7.
Figure 7.
Rebound of C1 unit activity, splanchnic SNA, and BP after RVLM illumination. AC, Effect of unilateral (A) or bilateral (B, C) RVLM illumination in rats previously injected with ArchT-expressing vectors. Gray bars represent the final portion of a 10 s illumination period. A, Postinhibitory rebound of a representative barosensitive neuron (presumptive C1 cell) that was silenced by light. Top trace, Raw signal. Bottom trace, Instantaneous action potential frequency (α-chloralose anesthetized rat). B, postinhibitory rebound of SNA (α-chloralose anesthetized rat). Top trace, Raw SNA. Bottom trace, Rectified and integrated signal (5 ms time constant). C, Postinhibitory rebound of BP in a conscious rat. Top, Raw signal. Bottom, Smoothed signal (1 s time constant). D, Average time course multiple experiments as in AC of postinhibitory rebound for neuronal unit activity (n = 6 units from 2 each PRSX8- or CaMKII-ArchT-injected rats, total 4 rats), rectified/integrated SNA (n = 7 CaMKII-ArchT injected rats), and MAP (n = 9 CaMKII-injected rats).
Figure 8.
Figure 8.
Light elicits large BP reductions in unanesthetized PRSx8-ArchT injected rats exposed to hypoxia but not hypercapnia. A, BP, HR, and respiratory flow (Flow) in a representative rat previously injected with PRSx8-ArchT. Bilateral RVLM illumination (gray bars) had no or small effects on BP and HR in a normoxic (control: 21% FiO2, 79% FiN2) or hypercapnic environment (21% FiO2, 6% FiCO2, 73% FiN2), but produced a large hypotension and mild bradycardia in hypoxia (12% FiO2, 88% FiN2). The light reduced FR and VT during hypercapnia and normoxia, but had little effect on these parameters during hypoxia, as described previously for this viral vector injected in a similar location (Basting et al., 2015). B, MAP at rest (closed circles) and at the end of a 10 s illumination of the RVLM (open circles) in a cohort of seven rats exposed to various inhaled gas mixtures. CE, Corresponding values of HR, FR, and VT. Note, FiO2 level 21% and FiCO2 0% represent the same data points. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 9.
Figure 9.
Comparison of cardiorespiratory changes induced by light in rats injected with CaMKII-ArchT versus PRSx8-ArchT. AD, Average (n = 7 for PRSx8-ArchT and n = 8 for CaMKII-ArchT) effects of photoactivation on MAP (A), HR (B), FR (C), and VT (D) in rats injected with PRSx8-ArchT (black bars) and CaMKII-ArchT (gray bars) under varying FiO2 and FiCO2 levels. Note, FiO2 level 21% and FiCO2 0% represent the same data points. E, F, Plot of normalized changes in respiratory frequency (FR, filled circles) and normalized changes in MAP (open circles) elicited by ArchT activation in rats injected with PRSx8-ArchT (E, n = 7) or CaMKII-ArchT (F, n = 8). Note that, in the PRSx8-ArchT cohort, the hypotension was associated with a drop in FR, whereas, in the CaMKII-ArchT rats, the hypotension was associated with an increase in FR. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 10.
Figure 10.
Addition of CO2 reverses the effects of hypoxia on cardiovascular variables. A, Representative recording of BP, HR, and respiratory flow (Flow) from a rat previously injected with PRSx8-ArchT. Bilateral photoinhibition of RVLM neurons (gray bars) had little effect on BP and HR in control conditions (21% FiO2, 79% FiN2), but produced a large hypotension and bradycardia in hypoxia (10% FiO2, 90% FiN2). Addition of CO2 to the hypoxic mixture (10% FiO2, 3% FiCO2, 87% FiN2) reduced the hypotension and bradycardia and restored some breathing inhibition. BE, Data for all experimental animals (n = 7): MAP (B), HR (C), FR (D), and VT (E). Filled circles are resting values; open circles are values during bilateral inhibition with ArchT. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 11.
Figure 11.
Time-dependent contribution of C1 neurons to BP after SAD. A, effect of bilateral RVLM illumination on BP and HR 24 h before (−1 d), 1 d after, or 20 d after SAD in a resting unanesthetized rat previously injected with PRSx8-ArchT. The light had very little effect before and 20 d after SAD, but produced a large hypotension the day after this surgery. B, C, Group data for denervated rats (n = 3). MAP during 10 s preceding light delivery (closed circles) and during the last 2 s of the 10 s light pulse (open circles). The values are the mean of at least five trials per time point for each rat. C, HR recorded during the same period of time as in B. D, E, MAP and HR in sham-operated rats (n = 3) subjected to the same protocol. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 12.
Figure 12.
Hemodynamic changes elicited by RVLM inhibition in rats injected with PRSx8-ArchT vector: comparison between SADand sham-operated animals. A, Before surgery, ArchT photoactivation produced the same degree of hypotension at rest or under isoflurane anesthesia in the SAD cohort (magenta bars; n = 3) and the sham-operated controls (black bars; n = 3). B, SAD and sham-operated rats had the same number of ArchT-transduced C1 RVLM neurons. C, BP variability 1 d before (blue), 1 d after (magenta), and 20 d after surgery (green) in a representative SAD rat (left) and a sham-operated rat (right). D, Maximum hypotension elicited by 10 s ArchT photoactivation before and after surgery in SAD (magenta bars) and sham-operated rats (black bars; n = 3 for each group). E, BP variability (reported as SD) before and after SAD (magenta bars) or sham surgery (black bars). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 13.
Figure 13.
sBRS is increased by hypoxia and, to a lesser degree, by hypercapnia. A, Example of one baroreflex “sequence” selected in a conscious rat. Arrows indicate BP pulses that were selected for measurement of sBRS (five contiguous pulses during which systolic BP and SBP increased and PI also increased at the indicated delays). B, sequences collected before (pre-SAD; black) and after (post-SAD, magenta) SAD in a representative animal. The slope of the average regression line is greatly reduced after SAD denoting baroreflex impairment. C, Mean sBRS was significantly reduced after SAD surgery (n = 3, magenta bars), but unchanged after sham operation (n = 3, black bars). The number of sequences per 1000 heart beats (n/1000 beats) was not significantly different between the groups. D, Sequences observed under normoxia at rest (control, black), hypoxia (blue), and hypercapnia (green) in one rat. The sBRS value for each animal in each condition is the slope of the regression line (thick darker lines). In this example, there are fewer sequences for hypoxia (n = 9) than control (n = 22) or hypercapnia (n = 27); however, this is mostly due to a shorter exposure time to hypoxia. The number of sequences per 1000 heart beats is similar between these conditions for this rat (4.8, 3.1, and 6.8 for control, hypoxia, and hypercapnia, respectively). EH, Average (n = 23) sBRS (E) as determined by the “sequence” method during control (21% FiO2), hypoxic (12% FiO2), and hypercapnic (6% FiCO2) conditions. E, sBRS was increased during hypoxia and hypercapnia (n = 23). In hypoxia, there were small, significant reductions in the number of sequences observed (n/1000 beats, F), the change in SBP over a sequence (change in SBP, G), and the number of beats in observed sequences (beats per sequence, H). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

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