Activation of Arch in Dbx1 preBötC neurons in freely behaving awake mice.

<p><b>A</b>, Airflow, V_T, MV, and <i>f</i>_R plotted continuously during two consecutive applications of 2-s light pulses. Inspiratory airflow is plotted downward, which reflects whole-body plethysmography. <b>A</b>_<b>1</b>, <b>A</b>_<b>2</b>, Expanded airflow traces from A. Yellow bars indicate 589-nm light application. <b>B</b>_<b>1</b>, <b>B</b>_<b>2</b>, Cycle-triggered averages of airflow from each bout prior to (not highlighted) and during illumination (highlighted). Note that inspiratory airflow is attenuated, whereas expiratory airflow is not. Time calibrations are shown for each panel.</p

The respiratory effects of Arch-mediated photoinhibition.

<p>The left column shows effects in sedated animals. The right column shows effects in freely-behaving awake animals. Cyan symbols pertain to illumination of the preBötC whereas magenta symbols pertain to illumination of the dorsal medulla rostral to preBötC. Respiratory measurements include T_i (first row), MV (second row), V_T (third row), and <i>f</i>_R (fourth row). Control, light application, and recovery data are shown for all experimental subjects. Double asterisks refer to the probability of a type I statistical error with alpha < 0.01. Triple asterisks refer to the probability of a type I statistical error with alpha < 0.001. “n.s.” (i.e., not significant) refers to the probability of a type I statistical error with alpha > 0.05.</p

Light application to the dorsal medulla rostral to preBötC in freely behaving awake mice.

<p><b>A</b>, Airflow, V_T, MV, and <i>f</i>_R plotted continuously during two consecutive applications of 2-s light pulses. Inspiratory airflow is plotted downward, which reflects whole-body plethysmography. <b>A</b>_<b>1</b>, <b>A</b>_<b>2</b>, Expanded airflow traces from A. Yellow bars indicate 589-nm light application. <b>B</b>_<b>1</b>, <b>B</b>_<b>2</b>, Cycle-triggered averages of airflow from each bout prior to (not highlighted) and during illumination (highlighted). Time calibrations are shown for each panel.</p

Light application to the dorsal medulla rostral to preBötC in sedated mice.

<p><b>A</b>, Airflow, V_T, MV, and <i>f</i>_R plotted continuously during two consecutive applications of 2-s light pulses. Inspiratory airflow is plotted upward, which reflects nose-cone measurements. <b>A</b>_<b>1</b>, <b>A</b>₂, Expanded airflow traces from A. Yellow bars indicate 589-nm light application. <b>B</b>_<b>1</b>, <b>B</b>_<b>2</b>, Cycle-triggered averages of airflow from each bout prior to (not highlighted) and during illumination (highlighted). Time calibrations are shown for each panel.</p

Light activation of Arch-expressing Dbx1 preBötC neurons hyperpolarizes Dbx1 neurons and precludes respiratory rhythm.

<p><b>A</b>, Rostral slice surface of a P2 <i>Dbx1</i>^{<i>CreERT2</i>};Ai35D mouse showing Arch-GFP expression. Dotted box marks the preBötC. <b>B</b>, Dodt image of the slice in A showing location of the preBötC relative to known anatomical markers, the principal loop of the inferior olive (IOP_loop) and semicompact division of the nucleus ambiguus (scNA). The scale bar represents 150 μm and applies to both A and B. <b>C</b>, Inspiratory Dbx1 neuron visually identified by membrane-delimited EGFP expression (top), Dodt contrast microscopy (middle), and by dialysis of Alexa Fluor 568 introduced via the patch pipette solution after the onset of whole-cell recording (bottom). Scale bar represents 10 μm. <b>D</b>, Membrane potential trajectory of the neuron in C with synchronous XII output. TTX was applied at 1 μM. Voltage and time calibrations are shown <b>E</b>, A non-Dbx1 neuron lacking EGFP expression (top), identified in Dodt contrast microscopy and via Alexa Fluor 568 dialysis (bottom). Scale bar represents 10 μm. <b>F</b>, Membrane potential trajectory of the non-Dbx1 preBötC neuron in E with synchronous XII output. <b>G</b>, Membrane potential trajectory of a non-Dbx1 neuron with inspiratory modulation. Voltage calibration in D applies to F and G; separate time calibrations are shown. 589-nm light application is indicated by yellow bars in D, F, and G.</p

Arch-EGFP expresion and histology of fiber-optic implants in adult <i>Dbx1</i>^{<i>CreERT2</i>};Ai35D mice.

<p><b>A</b>, EGFP expression in 35 week-old <i>Dbx1</i>^{<i>CreERT2</i>};Ai35D mouse. Scale bar represents 500 μM. <b>A´</b>, Inset of boxed region in A showing an expanded view of the ventral region of the slice, which includes the preBötC. Scale bar represents 250 μM. <b>B</b>, Bright field image of a thionin-stained section adjacent to A. Scale bar represents 500 μM and applies to B, C, and D. <b>B´</b>, Inset of boxed region in B showing an expanded view of the ventral region of the slice, which shows visible markers that co-locate with the preBötC including the semicompact division of the nucleus ambiguus (scNA) and the principal loop of the inferior olive (IO_loop). Scale bar represents 250 μM. <b>C</b>, Bright field images of adjacent thionin-stained sections from an experimental mouse whose fiber-optics and ferrules targeted the preBötC. <b>D</b>, Bright field image of thionin-stained section from an experimental mouse whose fiber-optics and ferrules targeted medullary circuitry dorsal and rostral to the preBötC.</p