Gene discovery in genetically labeled single dopaminergic neurons of the retina

Abstract

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, immunocytochemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.

Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection

“…This is consistent with accumulating reports indicating that SMART-amplification can maintain relative expression levels of low and abundant cellular mRNA/cDNA (Herrler, 2000;Chen et al, 2001;Zhumabayeva et al, 2001Zhumabayeva et al, , 2004Seth et al, 2003;Wang et al, 2003;Gustincich et al, 2004;Rihl et al, 2004;Wellenreuther et al, 2004). In fact, our previous studies demonstrated that frequencies of Vβ families in SMART-derived cDNA were similar to those in conventional cDNA or anchored PCR-enriched cDNA when using a semi-quantitative method (Chen et al, 2001).…”

“…It would therefore be useful to be able to enrich limited amounts of mRNA/cDNA and employ the enriched cDNA for real-time quantitation of antigen-specific T cell clones in the limited amounts of clinical specimens. The SMART-based amplification technique has proven to be a useful method to enrich a limited amount of mRNA/cDNA for a large-scale quantitation of gene expression or arrays (Herrler, 2000;Chen et al, 2001;Zhumabayeva et al, 2001Zhumabayeva et al, , 2004Seth et al, 2003;Wang et al, 2003;Gustincich et al, 2004;Rihl et al, 2004;Wellenreuther et al, 2004). However, SMART-based real-time quantitation technology has not been validated for its utility in quantitating or measuring frequencies of antigen-specific T cell clones in small amounts of PBMC or tissue.…”

“…We also utilized the Altas Switch Mechanism At the 5′-end of Reverse Transcript (SMART)-derived cDNA to clone and sequence TCR in a MHC/peptide tetramer-reactive CD8 T cell population (Chen et al, 2001). SMART-based amplification of cDNA has proven to be a useful technique to enrich total cellular mRNA/cDNA from a low level of <50 ng to a large quantity (Herrler, 2000;Zhumabayeva et al, 2001Zhumabayeva et al, , 2004Seth et al, 2003;Wang et al, 2003;Gustincich et al, 2004;Rihl et al, 2004;Wellenreuther et al, 2004). This novel technique has made it possible to perform a large-scale quantitation of gene expression and arrays using a limited amount of clinical specimens (Zhumabayeva et al, 2001(Zhumabayeva et al, , 2004Gustincich et al, 2004;Rihl et al, 2004).…”

Histamine receptors in mammalian retinas

“…In rat retinas, dopamine release is lowest at night and highest during the day, even in constant darkness and in animals without photoreceptors, adult RCS/N-rdy rats homozygous for the rdy (retinal dystrophy) allele of photoreceptors (Doyle et al, 2002b). These rhythms might be maintained by melatonin release from the pineal gland (Doyle et al, 2002a) or by endogenous oscillations in the dopaminergic amacrine cells themselves (Gustincich et al, 2004;Witkovsky, 2004), but histaminergic retinopetal axons, which remained intact in these experiments, might also play an important role. Although HR1 receptors typically mediate excitatory effects, there are also known inhibitory effects (Brown et al, 2001).…”

Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons

“…To describe the precise anatomy of DA cells and record from them physiologically, we generated a line of transgenic mice in which dopaminergic neurons were labelled genetically by human placental alkaline phosphatase [11], developed a technique for single cell mRNA amplification and used cDNA screening to identify the transcripts that are components of DA cells' expression profile [12].…”