Neuropeptide Y ProtocolsAmbikaipakan Balasubramaniam Springer Science & Business Media, 2008 M02 5 - 246 pages 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. |
Contents
| 3 | |
A Gαi RNAAntisense Expression Strategy to Investigate | 10 |
Neuropeptide Y Y5 Receptor Expression | 53 |
Developing Transgenic Neuropeptide Y Rats | 73 |
Antisense Oligonucleotide Approach | 103 |
Pharmacological Characterization | 115 |
with Antisense Oligodeoxynucleotide Screening Strategy | 129 |
to Study the Actions of | 151 |
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agarose gel aliquots antisense ODNs approx assay autoclave binding buffer cDNA clones codon concentration containing CREB culture cyclophilin denature DEPC described in Subheading digestion diluted distilled H2O EDTA eggs ethanol expression filters food intake fragment G-protein-coupled receptors genomic DNA homogenizer human NPY Y5 hybridization solution hypothalamic incubate injection isolated labeled ligand medium membrane mg/mL MgCl2 microcentrifuge tube mouse mRNA NaCl neuropeptide Y receptor nonspecific Note NPY gene NPY receptor NPY transgenic NPY Y5 receptor nucleotide oligonucleotide overnight pancreatic polypeptide pellet peptide YY phage pipet plasmid plates polymerase prehybridization Prepare primers probe pronucleus protein radiolabeled radioligand rat NPY reaction receptor subtype restriction enzyme Resuspend riboprobes Rinse RNase room temperature saline sample screening Sigma Southern blot sterile stored at 20°C targeting tion tissue transfected transfer transgene transgenic rats Tris-HCl vector wash µg/mL