Figure 1.

Cre-dependent expression of channelrhodopsin or ZsGreen in neurons of the Mesencephalic Locomotor Region (MLR) expressing the vesicular glutamate transporter 2 (Vglut2). (A) For anatomical experiments, homozygous mice expressing Cre-recombinase under the control of the Vglut2 promoter (Vglut2-Cre, see “Materials and Methods” section) were crossed with homozygous mice with ZsGreen preceded by a STOP cassette flanked by loxP sites preventing ZsGreen expression. In the resulting heterozygous mice (Vglut2-ZsGreen), if Vglut2 is expressed during cell lifetime, Cre-dependent recombination removes the STOP cassette, allowing permanent expression of ZsGreen under control of the CAG promoter. (B) For optogenetic experiments, mice homozygous for Vglut2-Cre were crossed with mice homozygous for channelrhodopsin (ChR2) and enhanced yellow fluorescent protein (EYFP) preceded by a STOP cassette flanked by loxP sites preventing their expression. In the resulting heterozygous mice (Vglut2-ChR2-EYFP), if Vglut2 is expressed during cell lifetime, Cre-dependent recombination removes the STOP cassette, allowing permanent expression of ChR2-EYFP under control of the CAG promoter. (C) Photomicrographs of transversal brain slices from Vglut2-ZsGreen mice at the MLR level. (D) Schematic representation of a brain slice at the MLR level. (E) Higher magnification of the brain slice in (C) at the level of the CnF. (F,G) Epifluorescent images taken in the CnF, showing that cells expressing ZsGreen (green) are immunopositive for the neuronal marker NeuN (red). (H) Whole-cell patch-clamp recording of a neuron recorded in a brainstem slice of a Vglut2-ChR2-EYFP mouse at the level of the MLR. The neuron spikes an action potential at short latency in response to a 10 ms blue light pulse. CnF, cuneiform nucleus; IC, inferior colliculus; PAG, periaqueductal gray; PPN, pedunculopontine nucleus.