Slc6a9 is distributed in glial cells and neurons across several nervous system regions, whereas Slc6a5 is more restricted to neurons in the caudal brain

The glycinergic system constitutes a main source of inhibitory regulation in the central nervous system. Glycine transporters (GLYT1 and GLYT2), encoded by Slc6a9 and Slc6a5, respectively, are responsible for glycine reuptake and clearance from the synaptic cleft, thereby maintaining neurotransmitter homeostasis. Emerging evidence from pharmacological and mechanistic studies has highlighted GLYTs as promising therapeutic targets for psychiatric disorders and persistent pain. Nevertheless, data on anatomical and cellular distribution of GLYTs and sex-dependent differences in GLYT expression remain limited. To address this gap, the aim of this study was to examine the Slc6a9 and Slc6a5 mRNA expression across mouse brain regions and peripheral organs using three complementary approaches focusing on mRNA expression: re-analysis of single-cell RNA sequencing data, quantitative RT-PCR, and RNAscope. Both genes were detected in multiple brain regions, with Slc6a9 exhibiting a broader distribution in both glial cells and neurons, while Slc6a5 was more restricted to neurons. Sex-dependent differences were detected for Slc6a9 in the amygdala and thalamus, liver, intestine, spleen, kidney and genitalia using quantitative RT-PCR, and for Slc6a5 in the cortex, striatum, hippocampus, and spinal cord using quantitative RT-PCR. Spatial analysis of the glycine transporters showed that Slc6a9 can be found in several brain regions spanning the rostral to the caudal axis, in both glial cells and neurons, while Slc6a5 was more restricted to the caudal brain regions. In general, in regions where differences were detected using quantitative RT-PCR, higher expression levels were observed in male mice. Moreover, Slc6a9 expression was found to occur in both glial cells, such as astrocytes, oligodendrocytes and ependymal cells, as well as both excitatory and inhibitory neurons, while Slc6a5 mainly occurred in inhibitory neurons. These findings provide novel insights into the spatial and sex-dependent expression of glycine transporters.