Photostimulation of Phox2b medullary neurons activates cardiorespiratory function in conscious rats

Abstract

Rationale: Hypoventilation is typically treated with positive pressure ventilation or, in extreme cases, by phrenic nerve stimulation. This preclinical study explores whether direct stimulation of central chemoreceptors could be used as an alternative method to stimulate breathing.

Objectives: To determine whether activation of the retrotrapezoid nucleus (RTN), which is located in the rostral ventrolateral medulla (RVLM), stimulates breathing with appropriate selectivity.

Methods: A lentivirus was used to induce expression of the photoactivatable cationic channel channelrhodopsin-2 (ChR2) by RVLM Phox2b-containing neurons, a population that consists of central chemoreceptors (the ccRTN neurons) and blood pressure (BP)-regulating neurons (the C1 cells). The transfected neurons were activated with pulses of laser light. Respiratory effects were measured by plethysmography or diaphragmatic EMG recording and cardiovascular effects by monitoring BP, renal sympathetic nerve discharge, and the baroreflex.

Measurements and main results: The RVLM contained 600 to 900 ChR2-transfected neurons (63% C1, 37% ccRTN). RVLM photostimulation significantly increased breathing rate (+42%), tidal volume (21%), minute volume (68%), and peak expiratory flow (48%). Photostimulation increased diaphragm EMG amplitude (19%) and frequency (21%). Photostimulation increased BP (4 mmHg) and renal sympathetic nerve discharge (43%) while decreasing heart rate (15 bpm).

Conclusions: Photostimulation of ChR2-transfected RVLM Phox2b neurons produces a vigorous stimulation of breathing accompanied by a small sympathetically mediated increase in BP. These results demonstrate that breathing can be relatively selectively activated in resting unanesthetized mammals via optogenetic manipulation of RVLM neurons presumed to be central chemoreceptors. This methodology could perhaps be used in the future to enhance respiration in humans.

Figure 1.

Experimental design. * Indicates the fraction of rats in which photostimulation increased breathing or renal sympathetic nerve discharge (SND). & Indicates group of eight rats in which optrode placement was always correct, but only four rats had normal numbers of channelrhodopsin-2 (ChR2)-transfected ccRTN neurons. Photostimulation activated breathing only in the latter rats. # Indicates fraction of rats in which the optrode was correctly placed in relation to the ChR2-transfected cells. The four rats with misplaced optrodes had no response to photostimulation. BP = blood pressure.

Figure 2.

Photostimulation of channelrhodopsin-2–transfected rostral ventrolateral medulla neurons activates breathing: representative plethysmography example. (A) Original trace showing the effect of a 1-minute period of photostimulation (20 Hz, 10-ms pulses, 12 mW) on inspiratory and expiratory flow in a conscious, resting rat. (B) Excerpt from A showing the initial phase of the breathing response. (C) Excerpt from A showing the end of the photostimulation period. Note that the breathing rate and flows were smaller toward the end of the stimulation period than at the beginning. Note also that the frequency and flows were reduced below the prestimulus baseline after interruption of the photostimulation. (D) Change in minute volume caused by the stimulus. (E) Change in tidal volume. (F) Change in breathing frequency. (G) Change in other plethysmography parameters (from top to bottom: inspiratory duration [Ti], expiratory duration [Te], expiratory relaxation time [RT], peak inspiratory flow, and peak expiratory flow).

Figure 3.

Photostimulation of channelrhodopsin-2–transfected rostral ventrolateral medulla neurons activates diaphragmatic activity in conscious rats: representative example. Top trace: rectified and integrated diaphragmatic EMG with amplitude normalized to 1 during resting period. Bottom trace: breathing frequency. Photostimulation was done for 30 seconds with 10-ms light pulses delivered at 20 Hz.

Figure 4.

Photostimulation of channelrhodopsin-2–transfected rostral ventrolateral medulla neurons activates renal sympathetic nerve activity and blood pressure in conscious rats. (A) Representative example. The traces from top to bottom are blood pressure (descending aorta, AP), heart rate (HR), integrated renal sympathetic nerve discharge (iSND; rectified and integrated with 2-s time constant; trace intentionally moved by 2 s to the right to correct for integration time and proper alignment with the other traces), and raw renal SND (100–3,000 Hz). Photostimulation was applied for 30 s (gray area) with 10-ms light pulses delivered at 20 Hz. (B) Average effect of photostimulation on blood pressure (n = 8). (C) Average effect of photostimulation on renal SND (n = 8). (D) Average effect of photostimulation on heart rate. * P < 0.05; ** P < 0.01. MAP = mean arterial pressure.

Figure 5.

Low-frequency photostimulation of channelrhodopsin-2–transfected rostral ventrolateral medulla neurons evokes large amplitude response in renal sympathetic nerve discharge (SND). (A) Representative example of the effect of 10-ms light pulses, applied every 10 seconds, on renal SND and blood pressure (BP). The far right of the trace shows an expanded trace of the same stimulus. (B) A laser pulse-triggered waveform average of a representative case. This case shows that after the initial burst, renal SND falls below baseline before slowly returning to prestimulus levels. (C) Paired-pulse paradigm. Each trace represents the evoked responses caused by a pair of 10-ms light pulses delivered at a fixed interpulse interval (S1 and S2). Small interpulse intervals (top trace) cause the second evoked response to be dramatically reduced. This reduction becomes smaller as the interpulse interval is increased (following three traces). The bottom trace illustrates that the evoked responses are abolished after administration of the ganglionic blocker chlorisondamine. (D) Attenuation of the second evoked response as a function of the interval between the pulses (eight rats; the asterisk indicates that the second evoked response of each pair was significantly smaller than the first).

Figure 6.

Photostimulation of channelrhodopsin-2–transfected rostral ventrolateral medulla (RVLM) neurons increases the range of the sympathetic baroreflex. (A) Second-by-second relationship between mean blood pressure and renal SND during the administration of sodium nitroprusside followed by administration of phenylephrine. The solid circles show the relationship at rest and the open circles show the relationship during photostimulation of the RVLM (20 Hz, 10-ms pulses). The best fit logistic curves are also shown. (B) Average baroreflex logistic curve for eight rats at rest and during photostimulation of the RVLM. Asterisk indicates that the lower plateau (maximum sympathetic nerve discharge [SND] observed when the baroreceptors are unloaded) was significantly (P < 0.05) elevated by RVLM photostimulation. (C) Average baroreflex gain plotted as a function of the mean blood pressure (MBP) for eight rats. The maximum gain was not significantly greater during photostimulation.

Figure 7.

Examples of transfected catecholaminergic and noncatecholaminergic Phox2b-positive neurons in the rostral ventrolateral medulla (RVLM). Left column illustrates the region immediately caudal to the facial motor nucleus. Almost all transfected cells (mCherry positive, red) have a Phox2b-immunoreactive nucleus (green) and most (small white arrows) contain tyrosine hydroxylase (TH, blue). The large white arrow points to a Phox2b-positive neuron that is transfected (mCherry positive) but does not contain TH. Right column illustrates the region immediately under the caudal end of the facial motor nucleus (the RTN). All transfected neurons in this region have a Phox2b-positive nucleus but they are devoid of TH (ccRTN neurons, white arrowheads). Rat brain tissue was cut into 30-μm sections through the RVLM and processed for immunohistochemistry as described in the Methods section. The scale bar at lower right (50 μm) applies to all panels.

Figure 8.

Distribution of channelrhodopsin-2–transfected catecholaminergic (C1) and noncatecholaminergic (ccRTN) neurons in the rostral ventrolateral medulla (RVLM). Mean numbers of channelrhodopsin-2 (ChR2)-expressing neurons counted on the left side of the RVLM in a one-in-six series of 30-μm coronal sections from 15 rat brains. Rats had been injected with ChR2-mCherry lentivirus and subjected to physiological experiments described in the current study. Cell numbers are plotted against the relative rostrocaudal position of the coronal section relative to bregma. These levels were determined relative to the landmark of the most caudal end of the facial motor nucleus designated as 11.6-mm caudal to bregma after the atlas of Paxinos and Watson (54). Error bars represent the SEM. Note that the bulk of the ChR2-transfected C1 cells (positive for both mCherry and tyrosine hydroxylase [TH], open circles, dotted line) are slightly caudal to the peak of the ccRTN neurons (positive for mCherry, negative for TH; filled circles, solid line), but the two populations overlap considerably.

Figure 9.

Optrode placement in two representative cases Photomicrographs showing the maximum optrode penetration in two cases with robust physiological responses to laser light stimulation. (A, B) Transverse sections showing mCherry fluorescent cells and processes (denoting the presence of channelrhodopsin-2). (A) Approximately 11.24 mm caudal to bregma (i.e., under the caudal third of the facial motor nucleus). (B) Approximately 11.6 mm caudal to bregma (i.e., under the very caudal end of the facial motor nucleus). The scale bar in B (250 μm) applies to both panels.