Figure 3. GABAergic neurons in the male.
(A–C) Anti-GABA staining of adult males in the head and tail in different optical sections. (Scale bar = 10 μm) (A) In adult male head, same neurons as in the hermaphrodite head are GABA-positive. (B) Seven male-specific neurons are GABA-stained: EF1-4, R2A, R6A and R9B (white arrows; two optical section with green fluorescent secondary antibody in left two panels with ray neuron staining being background and red fluorescent secondary antibody in upper right panels with ray neuron showing no background staining). (C) In unc-47 mutants, GABA staining in R2A, R6A and R9B persists, indicating that these neurons do not acquire GABA via secretion from other neurons. In addition, we observe GABA staining in an extra ray neuron, R3, that we cannot detect in wild-type animals. (D) Reporter gene expression in adult male tail. Left panel: unc-25 reporter, other panel shows red fosmid reporter (nucleus) and green anti-GABA staining. Reporter constructs as shown in Figure 2. EF1-4 expressed unc-25/GAD, unc-47/VGAT and snf-11/GAT. unc-47/VGAT is expressed in R6A but not in R2A or R9B. White arrow indicates GABA stained cells colocalizing with the reporter. snf-11/GAT is present in the male VD12 but not in the hermaphrodite. unc-25/GAD transcriptional reporter shows the same expression pattern as the gfp knock-in allele in the male tail (Scale bar = 10 μm).