Fig. 6

que mutants contain a reduced concentration of glutamate in the brain. (A–F) Cross-sectional views of the hindbrain of 96 hpf larvae are shown. Immunohistochemistry using antibodies against acetylated tubulin, which predominantly labels axon tracts, reveals the overall structure of the brain and demonstrates tissue penetration of the antibodies. Staining using an antibody against L-glutamate illustrates the distribution of this neurotransmitter. (A) Labeling with the anti-acetylated tubulin reveals the axon tracts and overall structure of the hindbrain of wild-type larvae. (B) The hindbrain of wild-type larvae contains a broad distribution of L-glutamate. (C) The merged images show several L-glutamate-positive cells surrounded by anti-acetylated tubulin labeling. (D) The overall structure of the hindbrain of que mutants revealed by anti-acetylated tubulin appears similar to the hindbrain wild-type larvae. (E) The fluorescence intensity of labeling with the L-glutamate antibody is greatly reduced compared with wild type when imaged using the same microscope settings. However, increasing the gain of the confocal microscope shows more faint L-glutamate staining (inset). (F) The merged images show little L-glutamate staining compared with acetylated tubulin labeling. (G) The graph shows a significant reduction in L-glutamate staining intensity in que mutants normalized to acetylated tubulin staining. The fluorescent intensity values are the analog-to-digital converter values of the entire frame (n=5 embryos, 12 sections, **P<0.01). Very similar results were obtained when a region of interest was selected to encompass a smaller, designated portion of the brain.