IMS shows GABA ion is enriched in the endosteal region of the BM. (A-C) IMS of WT bone sections (n = 2 mice). (A) “Bone regions imaged” shows slide with 3 regions selected for IMS analysis (1-3), in sum they represent an entire femur slice. The slide is spotted with commercial GABA standards. (B) Left to right, Optical images of captured regions, bone overlay of mass filter on optical image (to confirm localization to endosteum), mass filter (black background for increased visible intensity), and GABA standards (concentrations from 1 µM to 10 mM), for m/z 104 (adjusted intensity range between 7% [dark blue] and 82% [white]). (C) As in panel B, for m/z 86 (adjusted intensity range between 8% [dark blue] and 100% [white]). GABA standards show limit of detection is between 100 µM and 1 mM. (D) Detail showing endosteal enrichment of GABA signal in mass filter and bone overlay for m/z 86. (E) Quantification of IMS of femur sections from WT and Rag1^−/− mice show a significant decrease of GABA ion in the Rag1^−/− bones (P < .001). Error bars indicate standard deviation of data sets based on 4 measurements: 2 biological samples × 2 technical replicates per biological sample (see also supplemental Figure 2).