Immunohistochemical Staining of the Brain of the Tuatara Sphenodon punctatus

The tuatara (Sphenodon punctatus) is the only extant rhynchocephalian reptile and it appears to closely resemble fossil rhynchocephalians, implying retention of primitive rhynchocephalian traits. Because of their endangered status, however, there have been few modern studies of their brain. We have performed immunolabeling on the brain of a Sphenodon specimen for a variety of neurotransmitters and neuropeptide markers that have proven informative for characterizing brain regionalization in other species. These markers include leucine-enkephalin, the preproenkephalin-derived neuropeptide methionine-enkephalin-arg-phe, the preproenkephalin-derived neuropeptide peptide E, substance P, tyrosine hydroxylase, LANT6, neuropeptide Y, glutamic acid decarboxylase, serotonin, vasoactive intestinal polypeptide, luteinizing hormone releasing hormone, and cholecystokinin-8. Effective staining was obtained for all, except cholecystokinin-8, which for unknown reasons yielded only background perikaryal labeling that is nonetheless serviceable for characterizing cytoarchitecture. To show its value, we provide here an overview of the labeling obtained with each antibody, highlighting forebrain and midbrain, which shows remarkable resemblance to that reported in turtles. Images of the labeling will be made available at Brainmaps.org, and the slides will be banked at the Museum of Comparative Zoology at Harvard University, where they can be analyzed in greater depth by interested investigators.