We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1b, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X 7 , microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATPinduced shedding and IL-1b release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1b release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1b release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microgliato-neuron signalling pathway.

The authors report that the sequence of an RNA interference oligonucleotide for Mcm21R/CENP-O was incorrectly annotated in the Supplementary Materials and Methods to the article cited above. We had reported the oligonucleotide (ATATGAGTCTGGTCTCCTA) to comprise positions 959-977 of the coding sequence of Mcm21R (as measured from the start codon) whereas it actually lies between positions 884-902. This error arose because several sequences for Mcm21R cDNA versions existed in the NCBI database in 2006. Now that the sequence has been curated, we noticed our error. We apologize for any inconvenience caused.

Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB 1 ), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.

The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.

mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) ␣6 and ␤3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that ␣6-or ␤3-dependent nAChRs bind ␣-conotoxin MII (␣-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of ␤3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that ␤3-null mutation selectively reduced striatal ␣6* nAChR expression by 76% versus ␤3 ϩ/ϩ control. Parallel experiments showed a smaller reduction in both midbrain ␣3* and ␣6* nAChRs (34 and 42% versus ␤3 ϩ/ϩ control, respectively). Sedimentation coefficient determinations indicated that residual ␣6* nAChRs in ␤3 Ϫ/Ϫ striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified ␤3* nAChRs demonstrated that almost all wildtype striatal ␤3* nAChRs also contain ␣4, ␣6, and ␤2 subunits, although a small population of non-␤3 ␣6* nAChRs is also expressed. ␤3 subunit incorporation seemed to increase ␣4 participation in ␣6␤2* complexes.125 I-Epibatidine competition binding studies showed that the ␣-CtxMII affinity of ␣6* nAChRs from the striata of ␤3 Ϫ/Ϫ mice was similar to those isolated from ␤3 ϩ/ϩ animals. Together, the results of these experiments show that the ␤3 subunit is important for the correct assembly, stability and/or transport of ␣6* nAChRs in dopaminergic neurons and influences their subunit composition. However, ␤3 subunit expression is not essential for the expression of ␣6*, high-affinity ␣-CtxMII binding nAChRs.

Neuronal nicotinic receptors (nAChRs) are a heterogeneous family of ion channels differently expressed in the nervous system where, by responding to the endogenous neurotransmitter acetylcholine, they contribute to a wide range of brain activities and influence a number of physiological functions. Over recent years, the application of newly developed molecular and cellular biological techniques has made it possible to correlate the subunit composition of nAChRs with specific nicotine-elicited behaviours, and refine some of the in vivo physiological functions of nAChR subtypes. The major new findings are the widespread expression of nAChRs, outside the nervous system, their specific and complex organisation, and their relevance to normal brain function. Moreover, the combination of clinical and basic research has better defined the involvement of nAChRs in a growing number of nervous pathologies other than degenerative diseases. However, there are still only a limited number of nicotinic-specific drugs and, although some nicotinic agonists have an interesting pharmacology, their clinical use is limited by undesirable side effects. Some selective nicotinic ligands have recently been developed and used to explore the complexity of nAChR subtype structure and function in the expectation that they will become rational therapeutic alternatives in a number of neurodegenerative, neuropsychiatric and neurological disorders. In this review, we will discuss the molecular basis of brain nAChR structural and functional diversity mainly in pharmacological and biochemical terms, and summarise current knowledge concerning the newly discovered drugs used to classify the numerous receptor subtypes and treat the brain diseases in which nAChRs are involved.