Gene Discovery in Genetically Labeled Single Dopaminergic Neurons of the Retina

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 101; no. 14; pp. 5069 - 5074
• Main Authors: Gustincich, Stefano, Contini, Massimo, Gariboldi, Manuela, Puopolo, Michelino, Kadota, Koji, Bono, Hidemasa, LeMieux, Julianna, Walsh, Pamela, Carninci, Piero, Hayashizaki, Yoshihide, Okazaki, Yasushi, Raviola, Elio, Hubel, David H.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 06.04.2004; National Acad Sciences
• Subjects: Amacrine cells; Animals; Base Sequence; Biological Sciences; Cells; Complementary DNA; DNA Primers; DNA, Complementary; Dopamine - metabolism; Gene expression; Genes; Immunohistochemistry; Messenger RNA; Mice; Mice, Transgenic; Neurology; Neurons; Neurons - cytology; Neurons - metabolism; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polymerase chain reaction; Proteins; Receptors; Retina; Retina - cytology; Retina - metabolism; Ribonucleic acid; RNA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0400913101

In the retina, dopamine plays a central role in neural adaptation to light. Progress in the study of dopaminergic amacrine (DA) cells has been limited because they are very few (450 in each mouse retina, 0.005% of retinal neurons). Here, we applied transgenic technology, single-cell global mRNA amplification, and cDNA microarray screening to identify transcripts present in DA cells. To profile gene expression in single neurons, we developed a method (SMART7) that combines a PCR-based initital step (switching mechanism at the 5′ end of the RNA transcript or SMART) with T7 RNA polymerase amplification. Single-cell targets were synthesized from genetically labeled DA cells to screen the RIKEN 19k mouse cDNA microarrays. Seven hundred ninety-five transcripts were identified in DA cells at a high level of confidence, and expression of the most interesting genes was confirmed by immunocytochemistry. Twenty-one previously undescribed proteins were found in DA cells, including a chloride channel, receptors and other membrane glycoproteins, kinases, transcription factors, and secreted neuroactive molecules. Thirty-eight percent of transcripts were ESTs or coding for hypothetical proteins, suggesting that a large portion of the DA cell proteome is still uncharacterized. Because cryptochrome-1 mRNA was found in DA cells, immunocyto-chemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.