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 ...

(9), and Ca 2ϩ influx into lymphocytes (10). 5-HT 3 receptor activation mediates emetic and inflammatory responses (11) and may contribute to pain reception, anxiety, cognition, cranial motor neuron activity, modulation of affect, and the behavioral consequences of drug abuse (Refs. 12 and 13; but see Ref. 14).The 5-HT 3A receptor subunit shares structural similarities with members of the superfamily of ligand-gated ion channels (15) and is thought to be a pentameric protein (16, 17) with multiple agonist and allosteric ligand binding sites (2, 11). Both native and recombinant 5-HT 3 receptors reveal rapid and cooperative activation by agonists and desensitization to prolonged application of 5-HT (reviewed in Refs. 1, 2, and 6). With a few exceptions, ligand-gated channels require the association of more than one kind of homologous subunit for function, and subunit composition determines the pharmacological (18) and kinetic (e.g. desensitization (19,20)) profile of heteromeric receptors. While the 5-HT 3A subunit expressed in heterologous systems functions efficiently as homomers, different voltagedependence, desensitization, and pharmacological properties between recombinant and native 5-HT 3 receptors suggest that native 5-HT 3 receptors may exist as heteromers (3,(21)(22)(23)(24)(25)(26)(27). Although the 5-HT 3A gene encodes splice variants in mouse (28, 29) and guinea pig (30), most of the characteristics of these variants are similar (29,31,32), and the subtle pharmacological differences (22, 31, 32) cannot completely account for the differences observed between recombinant homomers and native 5-HT 3 receptors. In fact, co-expression of both splice variants in oocytes could not reproduce the responses observed in the cell line from which the splice variants were cloned (22). Biochemical studies on porcine brain have revealed the existence of at least four proteins (52-71 kDa) closely associated with the 5-HT 3A subunit that may represent antigenically distinct channel subunits (33).The cloning of a 5-HT 3B subunit that modified the pharmacological and single channel characteristics of the 5-HT 3A subunit when co-expressed in heterologous expression systems was recently reported (34). We extend this report and provide evidence for the co-existence of both 5-HT 3B and 5-HT 3A receptor subunits in native cells, a requirement for heteromeric association. We show that 5-HT 3B has no effect on the function of ␣2␤2, ␣3␤4, ␣4␤2, and ␣7 nicotinic ACh receptors. Furthermore, the characterization of the pharmacology and function of heteromeric receptors in Xenopus oocyte and mammalian expression systems reported here differs from that described (34), and these differences have important consequences for the function of heteromers in vivo.* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s) 1 The abbreviations used...

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

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CCK1R message can be conclusively detected and quantified in human pancreas cDNA by targeting the appropriate nucleotide sequence regions of this gene.