Gene expression profiling of the rat superior olivary complex using serial analysis of gene expression Alexander Koehl, 1 Nicole Schmidt, 1 Anne Rieger, 1 Sara M. Pilgram, 2 Ivica Letunic, 3 Peer Bork, 3 Florentina Soto, 2 Eckhard Friauf 1 and Hans Gerd Nothwang 1 1 Abteilung Tierphysiologie, Technische Universita ¨ t Kaiserslautern, Postfach 3049, 67653 Kaiserslautern, Germany 2 Abteilung Molekulare Biologie neuronaler Signale, Max-Planck-Institut fu ¨ r Experimentelle Medizin, Go ¨ ttingen, Germany 3 EMBL, Heidelberg, Germany Keywords: energy metabolism, gene expression, purinergic receptor, SAGE, Sprague–Dawley rats Abstract The superior olivary complex (SOC) is an auditory brainstem region that represents a favourable system to study rapid neurotransmission and the maturation of neuronal circuits. Here we performed serial analysis of gene expression (SAGE) on the SOC in 60-day-old Sprague–Dawley rats to identify genes specifically important for its function and to create a transcriptome reference for the subsequent identification of age-related or disease-related changes. Sequencing of 31 035 tags identified 10 473 different transcripts. Fifty-seven per cent of the unique tags with a count greater than four were statistically more highly represented in the SOC than in the hippocampus. Among them were genes encoding proteins involved in energy supply, the glutamate ⁄ glutamine shuttle, and myelination. Approximately 80 plasma membrane transporters, receptors, channels, and vesicular transporters were identified, and 25% of them displayed a significantly higher expression level in the SOC than in the hippocampus. Some of the plasma membrane proteins were not previously characterized in the SOC, e.g. the purinergic receptor subunit P2X 6 and the metabotropic GABA receptor Gpr51. Differential gene expression between SOC and hippocampus was confirmed using RNA in situ hybridization or immunohistochemistry. The extensive gene inventory presented here will alleviate the dissection of the molecular mechanisms underlying specific SOC functions and the comparison with other SAGE libraries from brain will ease the identification of promoters to generate region-specific transgenic animals. The analysis will be part of the publicly available database ID-GRAB. Introduction To a large extent, the pattern of expressed genes (i.e. the transcrip- tome) determines brain function. Knowledge about the transcriptome is therefore a fundamental requirement for in-depth understanding of neuronal processes. Genomics, through various concerted actions such as the genome projects in man, mouse, and rat, has provided information of almost the full complement of genes in these species (Lander et al., 2001; Waterston et al., 2002; Gibbs et al., 2004). These genomic tools, together with the invention of methods for large-scale gene expression analysis, now allow the identification of gene expression profiles to an unprecedented extent. One of the recently developed techniques for large-scale mRNA expression profiling is the serial analysis of gene expression (SAGE) (Velculescu et al., 1995). SAGE is a sequence-based method that does not require prior knowledge of the expressed genes. It is based on the reduction of each expressed transcript to a short (10–11 base-pair- long), yet representative, sequence (tag) defined by the last (most-3¢) occurrence of a certain restriction enzyme recognition site in the cDNA. In theory, ten base-pair-long tags can discriminate 4 10 ¼ 1048 576 sequences. The tags are concatenated into long molecules, and these concatemers yield information on multiple transcripts in a single sequence reaction. This considerably increases the efficacy compared to conventional expressed sequence tag (EST) projects. Furthermore, the number of times (counts) a particular tag is detected in a SAGE library provides a quantitative and digital measure of gene expression, which enables easy comparison between different SAGE libraries (Velculescu et al., 1995; Ruan et al., 2004). Studies of the mammalian brain transcriptome have to take into account that the brain is composed of a plethora of anatomically and physiologically distinct regions. One of the main challenges in neurobiology is therefore the identification of the specific transcrip- tomes of these different regions. To this end, we have started an extensive analysis of the mRNA complement of the superior olivary complex (SOC) using SAGE. The SOC is an essential processing centre in the mammalian auditory brainstem. It is the first binaural structure within the auditory pathway, i.e. it receives input from both ears. It consists of several nuclei, the major ones being the lateral superior olive (LSO), the medial superior olive (MSO), and the medial nucleus of the trapezoid body (MNTB). These nuclei are involved in sound localization by computing time or level differences between the two ears (Grothe, 2003). As accurate timing of auditory information is essential for this computation, auditory brainstem neurons display specializations that are not observed in any other sensory circuitry; very fast synaptic transmission and morphologically unusual synapses are striking examples (Oertel, 1999; Trussell, 1999; von Gersdorff & Borst, 2002). The important role in auditory information processing, together with the exquisite specializations, have made the SOC a favourable system to study neurotransmission in great detail (Trussell, 1999; Schneggenburger et al., 2002). The SOC is also widely used to study the structure and function of inhibitory neuronal circuits, as well Correspondence: Dr Hans Gerd Nothwang, as above. E-mail: nothwang@rhrk.uni-kl.de Received 9 June 2004, revised 8 September 2004, accepted 1 October 2004 European Journal of Neuroscience, Vol. 20, pp. 3244–3258, 2004 ª Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2004.03791.x