Sex-Specific Distribution of Glycine Transporter 2 in the Central Nervous System of Adult GlyT2-iCre-tdTomato Mouse Brain

Abstract

Glycine critically regulates locomotor and sensory processing, with its presynaptic glycine transporter 2 (GlyT2) serving as key therapeutic targets for modulating glycinergic neurotransmission. While GlyT2 inhibitors hold clinical promise, the specialization of spatially distributed GlyT2 neuronal populations across the brain remains incomplete. Here, we crossed GlyT2-iCre with Ai9 to generate GlyT2-iCre-tdTomato mice, enabling anatomical mapping and functional interrogation of GlyT2 circuits. Here, we generated GlyT2-iCre-tdTomato mice through crossbreeding GlyT2-iCre and Ai9 lines, enabling anatomical mapping and functional interrogation of GlyT2 circuits. Systematic quantification revealed sex- and region-specific neuronal distributions: non-estrus females exhibited significantly higher GlyT2-tdTomato neuron densities than males in 12 brain regions spanning thalamus (anterodorsal, anteroventral dorsomedial thalamic nucleus), midbrain (ventral periaqueductal gray, precommissural nucleus of the lateral lemniscus, superior and lateral parabrachial nucleus), and hindbrain (pontine nucleus, reticular oralis, reticulotegmental nucleus of the pons, rostromedial tegmental nucleus, solitary nucleus, interpolar part of spinal trigeminal nucleus), whereas males surpassed females in ventral posteromedial nuclei and lateral cerebellar regions. No sex differences were detected in 17 regions, including thalamic nuclei (anteromedial and ventral medial nucleus), posterior hypothalamus, substantia nigra, ventral nucleus of the lateral lemniscus, the nucleus of the central acoustic tract, ventral periolivary nucleus, paralemniscal nucleus, cerebellum, lateral periaqueductal gray, superior colliculus, inferior colliculus, medial parabrachial nucleus, and isthmic reticular formation. Spatial alignment with GlyT2mRNA data from the GlyT2-ISH atlas validated model specificity, establishing this resource as a foundation for investigating circuit-specific GlyT2 functions. These findings not only delineate organizational principles of glycinergic networks but also highlight sex as a critical variable in GlyT2 population architecture—a consideration essential for developing precision therapeutics targeting glycinergic dysfunction.