Substantial evidence indicates an association between clinical depression and altered immune function. Systemic administration of bacterial lipopolysaccharide (LPS) is commonly used to study inflammation-associated behavioral changes in rodents. In these experiments, we tested the hypothesis that peripheral immune activation leads to neuroinflammation and depressive-like behavior in mice. We report that systemic administration of LPS induced astrocyte activation in transgenic GFAP-luc mice and increased immunoreactivity against the microglial marker ionized calcium-binding adapter molecule 1 in the dentate gyrus of wild-type mice. Furthermore, LPS treatment caused a strong but transient increase in cytokine levels in the serum and brain. In addition to studying LPS-induced neuroinflammation, we tested whether sickness could be separated from depressive-like behavior by evaluating LPS-treated mice in a panel of behavioral paradigms. Our behavioral data indicate that systemic LPS administration caused sickness and mild depressive-like behavior. However, due to the overlapping time course and mild effects on depression-related behavior per se, it was not possible to separate sickness from depressive-like behavior in the present rodent model.

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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...

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