Acute itch has a clear beneficial purpose, directing our attention and physical response to specific areas of the skin to remove potentially harmful substances and parasites through scratching. However, millions suffer from persistent itch, where excessive scratching instead leads to skin injuries and sleep deprivation. Itch-inducing substances are detected by primary afferents in the skin, which relay the information to the dorsal horn of the spinal cord for further processing. Subsequently, ascending pathways transmit this information to the brain making us aware of the itch. Spinal glycinergic neurons play an important inhibitory role in the mouse central nervous system, as removal of these neurons results in a spontaneous itch behavior, and hypersensitivity to pruritogens injected in the skin. Glycine interacts with ligand-gated ion channels comprising alpha and beta subunits. We have previously characterized glycine receptor alpha 3 (Glra3) subunit-expressing neurons in the brain and spinal cord from female and male mice, revealing sex-dependent differences. However, the current understanding of how sensory information is transmitted from spinal cord to brain through projection neurons is limited within the field. This project aims to investigate Glra3 expression patterns in spinal cord projection neurons. Specifically, stereotaxic injections of a tracer in four different brain regions, the lateral parabrachial nuclei (LpB), the periaqueductal gray (PAG), the thalamus and the pontine nuclei, will elucidate potential overlaps between labelled projection neurons and Glra3 mRNA in the spinal cord using RNAscope analysis. Preliminary findings from the injections in LpB reveal successfully labelled projection neurons in the spinal cord.