Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains

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Abstract
Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether γ‐aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10−4 M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10−5 M serotonin was decreased by GABA; 10−5 M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10−4 M) partially inhibited palate shelf reorientation and that stimulated by 10−5 M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10−5 M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss‐Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water‐ and food‐starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10−5 M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administereted to the pregnant dam or added in embryo culture. Picrotoxin significantly antagonized these effects. Thus both GABA and diazepam inhibited palate reorientation. The SWV strain was more sensitive to both agents than the AJ; the posterior end was preferentially affected in SWV while the anterior was more sensitive in the AJ mouse. These results suggest that (1) GABA may function as an inhibitory neurotransmitter to regulate palate reorientation, (2) diazepam may cause cleft palate by mimicking GABA to inhibit shelf reorientation, and (3) these events are genetically regulated in inbred mouse strains.