Immunofluorescence was captured using a UltraVIEW VoX spinning disk confocal microscope, running Velocity software (v6.3.0, Perkin Elmer, Melbourne, Australia). In human motor cortex sections, nissl staining in conjunction with SMI32 labelling was used to demarcate cortical layers based on previously established methods [44 (link),45 (link)]. In mouse cortical sections, prior to image acquisition, a 20×/0.345 air objective (Nikon, New York, NY, USA) was used to identify grey matter of the primary motor cortex based on the appearance of anatomical landmarks, as described previously [46 ]. In mouse cortical sections, nissl staining was used to visualize neuronal nuclei and in conjunction with YFP-H labelling was used to demarcate cortical layers for receptor analysis. For NPY-Y1 receptor analysis, a 60×/0.0767 water objective (Nikon, New York, NY, USA) was used to capture a minimum of 6 z-stack images (0.4 μm intervals) of YFP-H-positive neurons (mouse cortex) and SMI32-positive (human motor neurons) across layers 5, 2/3 and 1.
To assess NPY-Y1 receptor expression in vitro, a 60×/0.0767 water objective (Nikon, New York, NY, USA) was used to capture z-stack images (0.2 μm intervals) of YFP-positive cortical neurons. Per individual embryo a minimum of 30 cells across triplicate coverslips were captured. All image acquisition was completed blinded to genotype.
To assess NPY-Y1 receptor expression in vitro, a 60×/0.0767 water objective (Nikon, New York, NY, USA) was used to capture z-stack images (0.2 μm intervals) of YFP-positive cortical neurons. Per individual embryo a minimum of 30 cells across triplicate coverslips were captured. All image acquisition was completed blinded to genotype.