The brain consists of various areas with anatomical features. Neurons communicate with one another via excitatory or inhibitory synaptic transmission. Altered abundance of neurotransmitters, including glutamate and gamma-aminobutyric acid (GABA), in specific brain regions is closely involved in severe neurological diseases, such as schizophrenia and obsessive-compulsive disorder. SCRAPPER, a ubiquitin E3 ligase, regulates synaptic transmission. Scrapper gene deficiency results in defective neurotransmission due to excessive secretion of neurotransmitters. The present study employed matrix-assisted laser desorption/ionization imaging mass spectrometry to analyze the abundance of amino acid neurotransmitters in Scrapper knockout (SCR-KO) mice. SCR-KO mice exhibited significantly increased glutamate levels in the isocortex (CTX), corpus callosum (CC), thalamus (TH), midbrain (MB), cerebellar cortex (CBX), and caudoputamen (CP) and increased GABA levels in the CTX, CC, TH, MB, CBX and hypothalamus (HY) compared with wild-type mice. These findings indicate that Scrapper deficiency leads to upregulated glutamate and GABA levels in multiple regions. Our results show a differential, region-specific effect of Scrapper on the abundance of glutamate and GABA.