GAD65/GAD67 double knockout mice exhibit intermediate severity in both cleft palate and omphalocele compared with GAD67 knockout and VGAT knockout mice

Abstract

Inhibitory neurotransmitters, γ-aminobutyric acid (GABA) and glycine, are transported into synaptic vesicles by the vesicular GABA transporter (VGAT). Glutamate decarboxylase (GAD) is a GABA-synthesizing enzyme and two isoforms of GAD, GAD65 and GAD67 are encoded by two independent genes. There was virtually no GABA content in GAD65/GAD67 double knockout (GADs DKO) mouse brains. Neither GABAergic nor glycinergic inhibitory postsynaptic currents were almost detected in VGAT knockout (KO) mouse cultured neurons and spinal cords. GAD67 KO and VGAT KO mice displayed developmental abnormalities, cleft palate and omphalocele, suggesting that GABAergic transmission is involved in palate and abdominal wall formations. However, the incidence and severity of both failures in GAD67 KO mice were lower and less than those in VGAT KO mice. These results raise the possibility that GABAergic transmission mediated by GAD65-produced GABA and/or glycinergic transmission contributed to both palate and abdominal wall formations. However, it still remains unclear whether GABAergic transmission mediated by GAD65 and glycinergic transmission contribute to those formations. Here, to answer these questions, we generated GADs DKO mice and compared the phenotypes of GADs DKO mice with those of GAD67 KO and VGAT KO mice. Our anatomical analyses demonstrated that the incidence of cleft palate and omphalocele in GAD67 KO mice was 65.8% and 58.9%, respectively, but the incidence of both phenotypes in GADs DKO and VGAT KO mice was 100%. The severity of cleft palate and omphalocele was evaluated by elevation of palate shelves and size and liver inclusion of omphalocele, respectively. We observed that the phenotypes of cleft palate and omphalocele in GADs DKO mice were more and less severe than those in GAD67 KO and VGAT KO mice, respectively. These results indicate the significant contribution of not only GAD65-mediated GABAergic but also glycinergic transmissions to both palate and abdominal wall formations.