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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The 5-HT1A and 5-HT1B receptors of serotonin play important roles as auto- and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5-HT1A) and in the globus pallidus and substantia nigra (5-HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5-HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane-associated 5-HT1A receptors could be estimated to be at least 30-40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5-HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5-HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5-HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto- and as heteroreceptors. The localization of 5-HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS.

Of all partners involved in G-protein coupled receptor (GPCR) signalling, the regulator of G-protein signalling (RGS) proteins are the only ones showing fast gene expression changes after various stimuli. These expression changes can offer feedback regulation to GPCR signalling as RGS accelerate the return of G-proteins to their inactive form and exert regulatory functions on intracellular effectors. However, it is not yet known which RGS regulate which receptor transduction pathways in the brain. To start to answer this question, we studied the influence of specific agonists and antagonists of the dopamine D1 and D2 receptors on the gene expression of the five most abundant RGS in the striatum: RGS2, RGS4, RGS8, RGS9 and RGS10. Only changes in RGS2 and RGS4 mRNA levels were observed. The D1 agonist SKF82958 and D2 antagonist haloperidol caused an up-regulation of RGS2 (+ 38.0% and + 41.6%, respectively). The D1 antagonist SCH23390 and D2 agonist quinpirole caused a down-regulation of RGS2 () 25.0% and ) 35.0%) and an up-regulation of RGS4 (+ 57.2% and + 52.5%). D1 and D2 receptors exert opposite effects on RGS2 expression, as they do on cAMP levels, suggesting a cAMP-mediated transcription of RGS2. This was confirmed by the unique induction of RGS2 (+ 111.1%) observed in the periventricular zone of the striatum after intracerebroventricular injection of forskolin. RGS4 was up-regulated only when RGS2 was down-regulated. This suggests that both RGS exert distinct functions. Considering the coupling of D1 and D2 receptors to the intracellular effector adenylate cyclase 5 (AC5) through their respective Ga subunits in the striatum, our data allow us to suggest that RGS2 regulates the D1/Gaolf/AC5 pathway and RGS4 the D2/Gao/AC5 pathway.