G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

Identification of genes preferentially expressed by microglia and upregulated during cuprizone-induced inflammation.

Microglia, monocytes, and peripheral macrophages share a common origin and many characteristics, but what distinguishes them from each other at the level of gene expression remains largely unknown. In this study, we compared the transcriptional profiles of freshly purified microglia, monocytes, and spleen macrophages using Affymetrix Mouse Genome arrays to identify genes predominantly expressed by microglia. Among tens of thousands of genes assayed, 127 potential candidates were found, including nine newly discovered genes encoding plasma membrane and extracellular proteins. In the brain, the latter were selectively expressed by microglia, as revealed by in situ hybridization. Three of them were confirmed to be exclusively (MSR2) or predominantly (GPR12, GPR34) expressed in the brain compared to the other tissues examined. Furthermore, all of these genes were upregulated in activated microglia after treatment with the demyelinating toxin cuprizone, suggesting that they play roles in neuroinflammation. In conclusion, this study reports the identification of new selective markers for microglia, which should prove useful not only to identify and isolate these cells, but also to better understand their distinctive properties.

Chemical characterization of newly generated neurons in the striatum of adult primates.

We recently demonstrated the existence of neurogenesis in the striatum of adult monkeys, but the number of striatal neurons generated under normal conditions was too small to establish their chemical phenotype. We therefore used brain-derived neurotrophic factor (BDNF), which promotes neuronal differentiation and survival and induces striatal neurogenesis in rodents, in an attempt to increase the number of newborn neurons in monkey striatum and facilitate their chemical characterization. An adenoviral vector (AdBDNF), encoding the human BDNF cDNA under the control of a strong promoter, was injected into the lateral ventricles (LVs) of adult squirrel monkeys, which were then treated with bromodeoxyuridine (BrdU). Two weeks after viral injection, numerous BrdU-positive cells were found within the striatum and many expressed microtubule-associated protein 2 (MAP-2) and neuronal nuclear protein (NeuN), two markers of mature neurons. Newborn neurons also expressed glutamic acid decarboxylase (GAD(65/67)), calbindin (CB) and dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), three markers of striatal projection neurons. We found no BrdU-positive neurons displaying the phenotype of striatal interneurons. Numerous BrdU-positive cells located near the subventricular zone (SVZ) coexpressed the migrating neuroblast markers polysialylated neural cell adhesion (PSA-NCAM) and doublecortin (DCX), suggesting that precursor cells could migrate from LVs to striatal parenchyma and develop a neuronal phenotype once they reach the striatum. However, many pairs of BrdU-positive nuclei were observed in the striatal parenchyma, suggesting that newborn neurons could also arise from resident progenitor cells. The present study demonstrates that a single injection of AdBDNF increases the number of newborn neurons into adult primate striatum and that newborn striatal neurons exhibit the chemical phenotype of medium-spiny projection neurons, which are specifically targeted in Huntington's disease.

Evidence of newly generated neurons in the human olfactory bulb.

The subventricular zone (SVZ) is known to be the major source of neural stem cells in the adult brain. In rodents and nonhuman primates, many neuroblasts generated in the SVZ migrate in chains along the rostral migratory stream (RMS) to populate the olfactory bulb (OB) with new granular and periglomerular interneurons. In order to know if such a phenomenon exists in the adult human brain, we applied single and double immunostaining procedures to olfactory bulbs obtained following brain necropsy in normal adult human subjects. Double immunofluorescence labelling with a confocal microscope served to visualize cells that express markers of proliferation and immature neuronal state as well as markers that are specific to olfactory interneurons. Newborn cells that express cell cycle proteins [Ki-67, proliferating cell nuclear antigen (PCNA)] were detected in the granular and glomerular layers (GLs) of the human olfactory bulb; these cells coexpressed markers of immature neuronal state, such as Doublecortin (DCX), NeuroD and Nestin. Numerous differentiating cells expressed molecular markers of early committed neurons [beta-tubulin class III (TuJ1)] and were also immunoreactive for glutamic acid decarboxylase (GAD), a marker of GABAergic neurons, or tyrosine hydroxylase (TH), a marker of dopaminergic neurons. Other early committed neurons expressed the calcium-binding proteins calretinin (CR) or parvalbumin (PV). These results provide strong evidence for the existence of adult neurogenesis in the human olfactory system. Despite its relatively small size compared to that in rodents and nonhuman primates, the olfactory bulb in humans appears to be populated, throughout life, by new granular and periglomerular neurons that express a wide variety of chemical phenotypes.

Novel aspects of the chemical anatomy of the striatum and its efferents projections.

This paper summarizes the results of some of our previous neuroanatomical and immunohistochemical studies on the organization of the striatum and its efferent projections in rodents, monkeys and humans. It also reports recent functional calcium-imaging data obtained in rat brain slices, as well as developmental results gathered with bromodeoxyuridine (BrdU) in monkeys. On one hand, single-axon tracing studies in rats and monkeys have revealed that the majority of striatofugal axon arborizes within most striatal target structures. In humans, SP-positive fibers were found to arborize in the two segments of the globus pallidus, where they were closely apposed to pallidal neurons that expressed the neurokinin-1 receptor (NK-1r). In agreement with such findings, calcium-imaging studies in rats have revealed that pallidal and nigral neurons are both responsive to SP. These findings suggest that the striatofugal projection system is much more widely distributed than previously thought and exerted a multifaceted effect upon its target sites. On the other hand, immunostaining studies in humans have shown the presence of several types of putative dopaminergic neurons intrinsic to the striatum. Furthermore, BrdU labeling experiments in monkeys have demonstrated that new neurons are generated throughout adult life in the striatum of normal monkeys and that their number can be markedly increased by the administration of neuronal growth factors. These findings open new therapeutic avenues for the treatment of neurodegenerative disorders that specifically affect the striatum.

The rostral migratory stream in adult squirrel monkeys: contribution of new neurons to the olfactory tubercle and involvement of the antiapoptotic protein Bcl-2.

The subventricular zone (SVZ) lying along the ependymal layer of lateral ventricle is known to generate neural progenitor cells throughout adulthood in specific areas of the mammalian brain. In rodents, the anterior region of the SVZ produces neuroblasts that migrate in chain toward the olfactory bulb along the so-called rostral migratory stream (RMS). In the present study, the organization of the RMS in a representative of New World primates - the squirrel monkey (Saimiri sciureus) - was studied by using bromodeoxyuridine (BrdU), a thymidine analogue that incorporates itself into the DNA of cells undergoing mitotic division. Double and triple immunofluorescence labelling with a confocal microscope served to visualize cells that expressed BrdU as well as molecular markers of neurogenesis. Numerous newborn (BrdU+) cells, many ensheated in glial (GFAP+) tubes, were scattered along the entire RMS in squirrel monkeys. Some of these BrdU+ cells expressed molecular markers for early committed neurons (TuJ1), postmitotic granular neuroblasts (TUC-4) or mature neurons (MAP-2, NeuN), and virtually all of them expressed the antiapoptotic protein Bcl-2. A significant number of BrdU+ cells were found to deviate from the main stream of the RMS. Instead of reaching the olfactory bulb, these cells migrated ventrally into the olfactory tubercle, where they expressed a mature neuronal phenotype (MAP-2). These findings reveal that the RMS in New World monkeys is mitotically robust and markedly extended and suggest that Bcl-2 might play a role in the survival and/or differentiation of newborn neurons destined to olfactory bulb and olfactory tubercle in primates.

Newly generated neurons in the amygdala and adjoining cortex of adult primates.

The subventricular zone remains mitotically active throughout life in rodents. Studies with tritiated thymidine, which is incorporated into the DNA of mitotic cells, have revealed that the rodent subventricular zone produces neuroblasts that migrate toward the olfactory bulb along the rostral migratory stream. A similar migratory stream has been documented in monkeys by using the thymidine analogue BrdUrd. The same approach showed that neurogenesis occurred in the dentate gyrus of adult primates, including humans. In the present study, experiments combining injections of BrdUrd and the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine, with the immunostaining for molecular markers of neurogenesis (polysialylated neural cell adhesion molecule, beta-tubulin-III, collapsin response mediator protein-4, neuronal nuclear protein) in New World (Saimiri sciureus) and Old World (Macaca fascicularis) monkeys have revealed that new neurons are produced in the amygdala, piriform cortex, and adjoining inferior temporal cortex in adult primates. These newborn neurons expressed the antiapoptotic protein Bcl-2 and formed a more-or-less continuous pathway that extended from the tip of the temporal ventricular horn to the deep portion of the temporal lobe. The production of newborn neurons in the amygdala, piriform cortex, and inferior temporal cortex seems to parallel the continuing addition of neurons in the olfactory bulb. These two concomitant phenomena may ensure structural stability and functional plasticity to the primate olfactory system and temporal lobe.

Proliferating cells can differentiate into neurons in the striatum of normal adult monkey.

In this study we used bromodeoxyuridine (BrdU), a thymidine analogue that is incorporated into the DNA of mitotic cells, to study the cytogenesis status of the striatum in normal, adult, squirrel monkeys (Saimiri sciureus). Three weeks following BrdU injection, numerous BrdU-labeled (+) cells were encountered within both the dorsal and the ventral striatum, including the nucleus accumbens. Their number ranged from 5 to 50 per 40 microm-thick section. These BrdU+ cells were more abundant medially than laterally and displayed a rostrocaudal-decreasing gradient in the caudate nucleus and putamen. Double-immunofluorescence confocal studies have revealed that about 5-10% of the BrdU+ striatal cells expressed the neuronal nuclear antigen (NeuN), a marker for mature neurons. These findings suggest that new neurons are produced throughout adult life in the striatum of normal, adult primates. This result raises the possibility of experimentally enhancing the recruitment of these newborn neurons as a means to alleviate the symptoms of neurodegenerative diseases that affect the striatum.