Constitutive expression of CCR2 chemokine receptor and inhibition by MCP-1/CCL2 of GABA-induced currents in spinal cord neurones.

In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.

Complex effects of stromal cell-derived factor-1 alpha on melanin-concentrating hormone neuron excitability.

Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.

Distribution, cellular localization and functional role of CCR2 chemokine receptors in adult rat brain.

Recent studies demonstrated that the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 and its receptor, CCR2, play important roles in various brain diseases. In this study, using quantitative autoradiography, we studied the pharmacological properties of [125l]MCP-1/CCL2 binding in rat brain and we clearly showed the distribution of CCR2 receptors in cerebral cortex, nucleus accumbens, striatum, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, mammillary bodies and raphe nuclei. Moreover, using double fluorescent immunohistochemistry, we showed that CCR2 receptors were constitutively expressed on neurons and astrocytes. Using RT-PCR methods, we demonstrated that CCR2 mRNA is present in various brain areas described above. Four hours after an acute intraperitoneal lipopolysaccharide injection, we showed that MCP-1/CCL2 binding was up-regulated in several brain structures; this effect took place on both CCR2B labelled neurons and astrocytes and to a lesser extent on activated microglia. To explore neurobiological function of CCR2, actimetric study was carried out. After intracerebroventricular injections of MCP-1/CCL2, we showed that motor activity was markedly decreased. Our results provide the first evidence for constitutive CCR2 receptor expression with precise neuroanatomical and cellular localizations in the brain, and its regulation during an inflammatory process, suggesting that MCP-1/CCL2 and CCR2 play important physiological and pathophysiological role(s) in the CNS.

First discrete autoradiographic distribution of aminopeptidase N in various structures of rat brain and spinal cord using the selective iodinated inhibitor [125I]RB 129.

The selective and potent aminopeptidase N inhibitor [125I]RB 129 has been used for the radioautographic localization of this enzyme in rat brain, spinal cord and intestine. Brain microvessels and intestine brush-border cells were shown to present a high concentration of aminopeptidase N. Moreover, a labeling of various brain structures was observed. A very high level of binding occurred in the meninges, choroid plexus, pineal gland, paraventricular nucleus and pituitary gland. Moderate to high labeling was also observed in the cortex, caudate-putamen, subthalamic nucleus, central periaqueductal gray, thalamus, as well as in the dorsal and ventral horn of the spinal cord, which are known to contain a high concentration of enkephalins, opioid receptors and neutral endopeptidase. This co-localization confirms the physiological implication of aminopeptidase N in the inactivation of enkephalins accounting for the requirement of dual inhibition of neutral endopeptidase and aminopeptidase N to observe highly significant morphine-like effects induced by the protected endogenous opioid peptides. Aminopeptidase N was also visualized in moderate to high levels in other brain structures such as the hippocampus, nucleus accumbens, substantia nigra, hypothalamus (dorsomedial and ventromedial nuclei), raphe nucleus, pontine nucleus, inferior olive, and in high concentration in the granular layer of cerebellum. In summary, aminopeptidase N has been visualized for the first time in numerous brain areas using the selective inhibitor [125I]RB 129. This iodinated probe could allow the ex vivo and in vivo localization of aminopeptidase N in various tissues to be investigated and may also be used to evaluate quantitative changes in aminopeptidase N expression in pathological situations. Aminopeptidase N, which preferably removes NH2-terminal neutral amino acids from peptides, has probably a host of substrates. Nevertheless, a certain in vivo selectivity could be achieved by the presence of the enzyme in structures where the peptide effector and its receptors are also co-localized.

Characterization and visualization of [125I] stromal cell-derived factor-1alpha binding to CXCR4 receptors in rat brain and human neuroblastoma cells.

Stromal cell-Derived Factor-1 (SDF-1alpha), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and modulates cell migration, differentiation, and proliferation. CXCR4 has been reported to be expressed in various tissues including brain. Moreover, CXCR4 has recently been shown to be one of the coreceptors for HIV-1 infection which could be implicated in HIV encephalitis. In the present study, the binding properties and autoradiographic distribution of [125I]SDF-1alpha binding to CXCR4 were characterized in the adult rat brain. SDF-1alpha binding and CXCR4 coupling system were also studied in human neuroblastoma cell line SK-N-SH. The binding of [125I]SDF-1alpha on rat brain sections was specific, time-dependent and reversible. The highest densities of CXCR4 were detected in the choroid plexus of the lateral and the dorsal third ventricle. Lower densities of [125I]SDF-1alpha binding sites were observed in various brain regions including cerebral cortex, anterior olfactory nuclei, hippocampal formation, thalamic nuclei, blood vessels and pituitary gland. In the choroid plexus, the IC(50) and K(d) of [125I]SDF-1alpha binding were respectively 0.6 nM and 0. 36 nM. Similar IC(50) values were obtained in other brain structures. A CXCR4 antagonist, bicyclam, competed with SDF-1alpha binding (30% inhibition at 10(-6) M). In SK-N-SH cells, [125I]SDF-1alpha bound to CXCR4 with a K(d) of 5.0 nM and a maximal binding capacity of 460 fmol/mg of protein. SDF-1alpha induced a rapid and transient intracellular calcium increase in SK-N-SH cells. These findings suggest that CXCR4 is highly expressed in some brain structures and have a regulatory role in the nervous system. The significance of this expression in the brain parenchyma and more specifically in the choroid plexus remains to be clarified in the normal as well as in the infected brain.