Selective Optogenetic Activation of Rostral Ventrolateral Medullary Catecholaminergic Neurons Produces Cardiorespiratory Stimulation in Conscious Mice

Anatomical distribution of the ChR2-expressing CA neurons and the location of the fiber optic tip. A1–C1, Fiber optic tip placements for the experiments identified above the drawings. Approximate distance from bregma shown in each section (top right). Scale bar: (in A1) A1–C1, 0.5 mm. A2–C2, Grouped rostrocaudal distribution of the RVLM-CA neurons (filled square, neurons ir for both mCherry and TH; open circles, total TH-ir neurons). Cells counted on left (injection) side only. AP with the arrow indicates the rostral tip and caudal extent of the area postrema. FN with the arrow indicates the caudal most location and rostral extension of the facial motor nucleus. Amb, Compact formation of the nucleus ambiguus; IO, inferior olive; pyr, pyramidal tract.

Selective activation of RVLM-CA neurons activates breathing in anesthetized DβH^Cre/0 mice. A, Representative recording of dEMG from an anesthetized mouse (bottom, raw signal; top, rectified and integrated signal, idEMG). B, Breathing activation produced in a representative anesthetized mouse by hypercapnia and stimulation of ChR2-transfected RVLM-CA neurons with laser light (20 Hz, 20 ms long light pulses delivered at the times represented by the gray columns). C1, Grouped data for the effect of hypercapnia (8% CO₂), photostimulation of RVLM-CA neurons at 20 Hz with 5 ms or 20 ms light pulses, and photostimulation of the cerebellum (Cb stim) on dEMG amp (N = 7; *p < 0.05). C2, As per C1 for EMG f_R (N = 7; *p < 0.05). C3, As per C1 for f_R × dEMG amp (N = 7; *p < 0.05).

Optogenetic activation of ChR2-expressing RVLM-CA neurons activates breathing in conscious DβH^Cre/0 mice. A, Plethysmography recording of the respiratory effects of photostimulation in an AAV2-DIO-EF1α-ChR2-mCherry-injected DβH^Cre/0 mouse (top, respiratory frequency (f_R); bottom, respiratory flow signal, upward deflection in waveform represents inspiration). B, C, Relationship between initial change in f_R and stimulation frequency at constant light pulse duration (2 ms) (B) and light pulse duration at constant stimulation frequency (20 Hz) (C) in experimental (ChR2-mCherry) and control mice (mCherry alone). ***p < 0.001 for resting versus stimulated in experimental mice by two-way ANOVA with multiple comparisons. ^#p < 0.05, ^##p < 0.01 for the interaction of ChR2-mcherry expression and stimulation in experimental versus control mice. D, Time course of the change in f_R during 30 s stimulation trials at 20, 10, and 5 Hz with a 5 ms pulse width (top to bottom traces, respectively) (N = 7). E, Two examples of photostimulus-triggered histograms plotting the onset of inspiration relative to light pulses delivered at low frequency designed to entrain respiratory rhythm. Both the raster plot and binned event counts (10 ms bins) indicate that single (left, 10 ms pulse at 2 Hz) and double (right, 5 ms pulses at 1.83 Hz) light pulses produced a clustering of inspiratory events shortly after the stimulus. Lower trace, Photostimulus-triggered average of respiratory flow during the entrainment protocol.

Changes in breathing frequency during stimulation of RVLM-CA neurons is differentially occluded by hypoxia and hypercapnia. A, Initial respiratory effects of photostimulation (5 ms pulse at 20 Hz for 30 s) and poststimulus hypoventilation in a ChR2-mCherry-expressing DβH^Cre/0 mouse during hyperoxia (100% O₂, trace 1), hyperoxic hypercapnia (3 and 6% CO₂ balanced in O₂, traces 2 and 3, respectively), and a different mouse during normoxia (21% O₂ balanced in N₂, trace 4) and poikilocapnic hypoxia (15 and 10% O₂ balanced in N₂, traces 5 and 6). Arrows identify bouts of poststimulus hyperventilation. Note that the period of hypoventilation following the offset of the stimulus under hyperoxic and normoxic conditions is largely abolished when respiratory drive is increased by activating the chemoreceptors. B, Group data of the effects of photostimulation on f_R (B1), V_T (B2), and MV (B3) under hyperoxic and hyperoxic hypercapnic conditions. N = 9 for each condition, *p < 0.05, **p < 0.01, ***p < 0.001 for resting versus stimulated condition. C, Group data of the effects of photostimulation on f_R (C1), V_T (C2), and MV (C3) under normoxic and hypoxic conditions. N = 7 for each condition *p < 0.05, **p < 0.01 for resting versus stimulated condition. D, X-Y plot of the average change in f_R during stimulation in relation to prestimulus f_R under each gas mixture presented in B1 and C1 (p = 0.0001 for difference between slopes, hypercapnia r² = 0.44, hypoxia r² = 0.73; error bars indicate 95% CI). E, Change in f_R caused by RVLM-CA neuron stimulation and a brief CO₂ challenge (15 s of 15% CO₂) during graded hypoxia, normalized to the response f_R under normoxic conditions. **p < 0.01, ***p < 0.001 for stimulation versus CO₂ condition.

Selective activation of RVLM-CA neurons increases cardiorespiratory coupling and autonomic activation in conscious DβH^Cre/0 mice. A, Cardiovascular and respiratory effects of RVLM-CA neuron stimulation (5 ms pulse at 20 Hz). BP, arterial blood pressure; HR, heart rate. B1, B2, Expanded traces of resting (B1) and stimulated (B2) segments in A. During stimulation a marked increase in the synchronicity of the cardiac and respiratory cycles (sinus arrhythmia) is manifested by increased beat-to-beat variability. Broken vertical lines mark onset of inspiration and highlight sinus arrhythmia. C, Phase histogram plot of occurrence of BP nadirs (representing cardiac contraction) relative to the respiratory cycle (triggered from the rising phase of inspiration) plotted from A. Note that while the respiratory cycle was shortened by stimulation, the phase duration of inspiration was unchanged (resting, 0–130°; stimulated, 0–137°). D–H, Effects of photostimulation (5 ms, 20 Hz, 30 s) on MBP and HR after intraperitoneal injection of saline (D), methyl-atropine (E), propranolol (F), methyl-atropine plus propranolol (G), and prazosin (H). (*Overlapping lines reflect significant differences: one line, p < 0.05; two lines p < 0.01; three lines p < 0.001 by Dunnett's post test between drug and saline treatment.)