Vs120 s1

For delineation of SOC nuclei and counting of cholinergic neurons, sections were imaged with a virtual slide microscope (VS120 S1, Olympus BX61VST, Olympus-Deutschland, Hamburg, Germany) at 10× magnification using the proprietary software dotSlide® (Olympus). All three colors of the secondary antibodies used for immunostaining were acquired sequentially and could be visualized separately or in overlay (Figure 2).
For counts of total neuron numbers in LSO, confocal optical sections were acquired with a Leica TCS SP5-2 confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) with a Plan Fl20x/0.70 NA objective for the MAP2 stain. Stacks of eight-bit grayscale images were obtained with an axial distance of 3 µm between optical sections each averaged from four successive scans. Finer details (Figure 3B) were taken with a Plan 63x/NA1.32 oil immersion objective. For each optical section the images of one or two fluorochromes were collected sequentially. RGB stacks, montages of RGB optical sections, and maximum-intensity projections were assembled into tables by using ImageJ 1.37k plugins (NIH, USA) and Photoshop (CS6, Adobe Systems, San Jose, CA, USA). Figure images were arranged using CorelDRAW X6 (Corel Corporation, Ottawa, ON, Canada).

A one year old female ferret weighing 620 g received a 2 µl injection of pAAV2.5-CaMKIIa-hChR2(H134R)-EYFP (PennCore) as anterograde tracer into left FC. Six months later the animal was perfused and the brain was cryoprotected, shock frozen and cut on a cryostat into 50 µm thick frontal sections into parallel series of which one was counterstained with neutral red. For overview images, combined brightfield and fluorescence images were taken with a Hamamatsu slide scanner 2.0HT (Institut de la Vision) (Figure 2e, left). For details, fluorescence images were taken with a virtual slide microscope (VS120 S1, Olympus BX61VST) at 10× magnification (Figure 2e, right). Anatomical structures were reconstructed in accord with the ferret brain atlas (Radtke-Schuller, 2018 (link)).