Figure 1. Effect of photostimulating Vglut2::Chr2-expressing embryonic retrotrapezoid nucleus (RTN) neurons on membrane potential and motor output.
(A) Cartoon of a ventral view of the hindbrain showing the position of nVII (dotted line), RTN (red outline), the preBötC and the C4 phrenic nerve roots. (B) Ventral view corresponding to the boxed area in A (anterior at top, lateral on the left) of the RTN (red outline) and nVII (stippled outline) in a Vglut2::ChR2-YFP embryo, labeled for the indicated markers. (C) Sagittal section, labeled for the indicated markers, showing the Phox2b+/YFP+/Islet1,2− cells ventral to nVII and optimally accessible to light. (D) Above, ventral view of YFP expression in the RTN region of an E14.5 Vglut2::ChR2-YFP embryo showing the three recorded and biocytin-filled RTN cells (left panel). A high magnification of the one in the boxed area is provided in the right panels showing biocytin and YFP labeling separately and the overlay. Below, membrane potential trajectory of the RTN cell shown at high magnification, featuring spontaneously rhythmic (stars) and light-evoked (blue bars) bursts of action potentials. (E) Left panel, photostimulation of the RTN region in E15.5 brainstem-spinal cord preparations from a Vglut2::ChR2-YFP embryo evokes Calcium Green-1 AM (ΔF/F) changes in the RTN and adjacent nVII. Right panel, integrated C4 recording in such a preparation showing spontaneous C4 bursts of activity (stars) and bursts evoked by photostimulation (blue bars). Note that the light pulses entrain the ongoing endogenous rhythm. (F) In the presence of DAMGO that depresses the excitability of the preBötC, but not of the RTN, photostimulation of the RTN region in E15.5 preparations still evokes ΔF/F changes in the RTN but not in nVII (left panel). Right panel, above, integrated C4 activity showing systematic failures of photostimulation to evoke C4 responses. Below, set of 10 superimposed C4 activity traces. (G) Same experiment in Vglut2::ChR2;Dbx1LacZ/LacZ mutants (n = 5) in which the preBötC but not the RTN is disrupted. The mutation completely abrogates light-evoked nVII and C4 responses.