Gene expression profiles of thousands of genes can now be examined en masse through cDNA and oligonucleotide microarrays 1-3 . Recently, studies have been reported that examined gene expression changes in yeast 4,5 , as well as in mammalian cell lines 6 , primary cells 7 and tissues 8 . However, present applications of microarray technology do not include the study of gene expression from individual cell types residing in a given tissue/organ (that is, in situ). Such studies would greatly facilitate our understanding of the complex interactions that exist in vivo between neighboring cell types in normal and disease states. We demonstrate here that gene expression profiles from adjacent cell types can be successfully obtained by integrating the technologies of laser capture microdissection 9 (LCM) and T7-based RNA amplification 10 with cDNA microarrays 11 . Neighboring small and large neurons are individually capturedTo demonstrate this integration of technologies, we examined the differential gene expression between large-and small-sized neurons in the dorsal root ganglia (DRG). In general, large DRG neurons are myelinated, fast-conducting and transmit mechanosensory information, whereas small neurons are unmyelinated, slow-conducting and transmit nociceptive information 12 . We chose this system because numerous differentially expressed genes (small versus large) have been reported, thus the success of this experiment could be assessed; and because many small and large neurons are adjacent to each other, thus we could test whether individual neurons can be cleanly captured. Large (diameter of >40 µm) and small (diameter <25 µm and with identified nuclei) neurons were cleanly and individually captured by LCM from sections (10 µm in thickness) of Nissl-stained rat DRG (Fig. 1). For this study, two sets of 1,000 large neurons and three sets of 1,000 small neurons were captured for cDNA microarray analysis. RNA amplification is reproducible between individual capturesRNA was extracted from each set of neurons and linearly amplified (independently) an estimated 10 6 -fold using T7 RNA polymerase. After being amplified, one fluorescently labeled probe was synthesized from an individually amplified RNA (aRNA), divided equally into three parts and hybridized in triplicate to a microarray ('chip') containing 477 cDNAs (see Methods for chip design) plus 30 cDNAs encoding plant genes (for the determination of non-specific nucleic acid hybridization). Expression in each neuronal set (called S1, S2 and S3 for small and L1 and L2 for large neurons) was thus monitored in triplicate, requiring a total of 15 microarrays. The quality of the microarray data is demonstrated by pseudocolor arrays, one resulting from hybridization to probes derived from neuronal set S1 and the other from neuronal set L1 (Fig. 2a). In Fig. 2a, the enlarged part of the chip shows some differences in fluorescence intensity (that is, expression levels) for particular cDNAs and demonstrates that spots containing the different cDNAs are relatively uniform ...

Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.

The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptoractivation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors. G protein-coupled receptors (GPCRs) have a superior success record as drug targets, which fueled the interest in the identification of novel GPCRs. As a consequence, reverse pharmacology (1), the process that leads from an orphan receptor to the identification of its endogenous ligand, already has yielded approximately 40 novel receptor͞ligand pairs (for a recent review see ref.2). In some cases, completely unknown hormones or neurotransmitters, such as nociceptin (3), prolactin-releasing peptide (4), apelin (5), and the orexins (6), were discovered.While cloning novel GPCRs by degenerate primer (PCR) we found a unique GPCR in a rat cortex cDNA preparation. Analysis of its sequence analysis by BLAST revealed that this receptor did not group within any of the GPCR families activated by a known ligand. The most closely related sequences-the sensory neuron-specific receptors (7) and the MAS-related gene (Mrg) receptors (8)-belong to families that contain only orphan receptors themselves. To characterize this additional receptor we mapped its tissue distribution and tried to identify its natural ligand. Materials and MethodsCloning and Expression of the Rat Adenine Receptor. The FastTrack 2.0 kit (Invitrogen) was used to isolate mRNA from rat brain cortex, which was then reverse-transcribed into cDNA with the SMART RACE (rapid amplification of cDNA ends) cDNA amplification kit (CLONTECH). The initial rat adenine receptor cDNA fragment was derived from a degenerate primer PCR containing primers complementary to the TM2 region (5Ј-AATCTGTTCCTGATGACGCTGGCGT-3Ј) and TM7 region (5Ј-GGTGGTTGAGGCAGCAATAGATGATGGGGTT-3Ј) (9). For the elongation of the PCR fragment to the full-length reading frame, the SMART RACE cDNA amplification kit was used. The full-length coding sequence (GenBank accession no. AJ311952) was subcloned into pcDNA3, and the resulting expression construct was used for transient and stable expression in mam...

No abstract