Neuropeptide Y Protocols

The observation that neuropeptide Y (NPY) is the most abundant peptide present in the mammalian nervous system and the finding that it elicits the most powerful orexigenic signal have led to active investigations of the properties of the NPY family of hormones, including peptide YY (PYY) and pancreatic polypeptide (PP). Nearly two decades of research have led to the identification of several NPY receptor subtypes and the development of useful receptor selective ligands. Moreover, these investigations have imp- cated NPY in the pathophysiology of a number of diseases, including feeding disorders, seizures, memory loss, anxiety, depression, and heart failure. Vigorous efforts are therefore continuing, not only to understand the bioche- cal aspects of NPY actions, but also toward developing NPY-based treatments for a variety of disorders. To facilitate these efforts, it was decided to produce the first handbook on NPY research techniques as part of the Methods in Molecular Biology Series. In compiling Neuropeptide Y Protocols, I have gathered contributions on techniques considered critical for the advancement of the NPY field from experts in various disciplines. Each chapter starts with a brief introduction, with Materials and Methods sections following. The latter sections are presented in an easy to follow step-by-step format. The last section of the chapter, Notes, highlights pitfalls and the maneuvers employed to overcome them. This information, not usually disseminated in standard research pub- cations, may prove extremely useful for investigators employing these te- niques in NYP research.

Part I. Cloning Techniques. Cloning Neuropeptide Tyrosine cDNA, Carolyn A. Worby. Human Y1/Y5 Receptor Gene Cluster: Isolation and Characterization, Herbert Herzog. Human Type 2 Neuropeptide Y Receptor Gene: Isolation and Characterization, David A. Ammar and Debra A. Thompson. Y4 Receptor in Different Species: Functional Expression and Binding, Ingrid Lundell, Magnus M. Berglund, and Dan Larhammar. Neuropeptide Y Y5 Receptor Expression, Eric M. Parker. Homology-Based Cloning Methods: Identification of the NPY Y2, Y4, and Y6 Receptors, Douglas J. MacNeil and David H. Weinberg. Part II. Production of Transgenic and Knock-Out Models. Developing Transgenic Neuropeptide Y Rats, Mieczyslaw Michalkiewicz and Teresa Michalkiewicz. Neuropeptide Y Y1 Receptor-Deficient Mice: Generation and Characterization, Thierry Pedrazzini and Josiane Seydoux. Part III. Antisense Technology. Antisense Oligonucleotide Approach to Study NPY-Mediated Feeding Signal Transduction, Sulaiman Sheriff, William T. Chance, and Ambikaipakan Balasubramaniam. A Gai RNA-Antisense Expression Strategy to Investigate Coupling of Peptide YY/Neuropeptide Y Receptor to Gai, Thierry Voisin, Anne-Marie Lorinet, Mathieu Goumain, and Marc Laburthe. NPY Y5 Receptor Subtype: Pharmacological Characterization with Antisense Oligodeoxynucleotide Screening Strategy, Andrea O. Schaffhauser, Alain Stricker-Krongrad, and Karl G. Hofbauer. NPY Antisense Oligodeoxynucleotides to Study the Actions of NPY, Pushpa S. Kalra and Satya P. Kalra. Part IV. Techniques to Study Tissue Distribution of mRNA. Localization of Y-Receptor Subtype mRNAs in Rat Brain by Digoxigenin Labeled In Situ Hybridization, Rachel Parker and Herbert Herzog. Central Y4 Receptor Distribution: Radioactive Ribonucleotide Probe In Situ Hybridization with In Vitro Receptor Autoradiography, Philip J. Larsen and Peter Kristensen. Combining Non-Isotopic Localization of NPY mRNA with Immunocytochemistry, Jennifer Lachey, Jennifer Profitt, Chad Foradori, and RandyJ. Seeley. Part V. Miscellaneous Techniques. Y Receptors Characterized by RT-PCR: Distribution in Rat Intestine, Mathieu Goumain, Thierry Voisin, and Marc Laburthe. Quantification of NPY mRNA by Ribonuclease Protection Assay, Abhiram Sahu. Radioligand Binding Studies: Pharmacological Profiles of Cloned Y-Receptor Subtypes, Karen E. McCrea and Herbert Herzog. Index.