Mutations in ric-3 (resistant to inhibitors of cholinesterase) suppress the neuronal degenerations caused by a gain of function mutation in the Caenorhabditis elegans DEG-3 acetylcholine receptor. RIC-3 is a novel protein with two transmembrane domains and extensive coiled-coil domains. It is expressed in both muscles and neurons, and the protein is concentrated within the cell bodies. We demonstrate that RIC-3 is required for the function of at least four nicotinic acetylcholine receptors. However, GABA and glutamate receptors expressed in the same cells are unaffected. In ric-3 mutants, the DEG-3 receptor accumulates in the cell body instead of in the cell processes. Moreover, co-expression of ric-3 in Xenopus laevis oocytes enhances the activity of the C.elegans DEG-3/DES-2 and of the rat a-7 acetylcholine receptors. Together, these data suggest that RIC-3 is speci®cally required for the maturation of acetylcholine receptors.

Unbiased methods to assess the firing activity of individual neurons in the neocortex have revealed that a large proportion of cells fire at extremely low rates (<0.1 Hz), both in their spontaneous and evoked activity. Thus, firing in neocortical networks appears to be dominated by a small population of highly active neurons. Here we use a fosGFP transgenic mouse to examine the properties of cells with a recent history of elevated activity. FosGFP-expressing layer 2/3 pyramidal cells fired at higher rates compared to fosGFP− neurons, both in vivo and in vitro. Elevated activity could be attributed to increased excitatory and decreased inhibitory drive to fosGFP+ neurons. Paired-cell recordings indicated that fosGFP+ neurons had a greater likelihood of being connected to each other. These findings indicate that highly active, interconnected neuronal ensembles are present in the neocortex and suggest these cells may play a role in the encoding of sensory information.

and the §Instituto de Neurociencias, Alicante, Spain In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat ␣7, and human ␣7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human ␣4␤2 and ␣3␤4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT 3 serotonin receptors, whereas it barely modified ␣1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo.Nicotinic acetylcholine receptors are widely expressed ligand-gated ion channels that mediate fast synaptic excitation and have additional roles including modulation of synaptic release (1). The nAChRs 1 are homomers or heteromers composed of five subunits. Each subunit traverses the membrane four times and is posttranslationally modified by both disulfide bond formation and glycosylation. Maturation of nAChRs, leading to production of fully assembled and functional receptors on the plasma membrane, is a complex, time-consuming, and poorly characterized process (2-4). Recently, we identified a Caenorhabditis elegans gene, ric-3, likely to be an important player in the maturation of nAChRs. In C. elegans, RIC-3 is required for cholinergic transmission mediated by nAChRs in neurons and in muscles but not for synaptic transmission mediated by other ligand-gated ion channels, even when these are expressed within the same cells as the nAChRs. RIC-3 activity is required within the cells that express the nAChRs and is likely to affect the processes of receptor folding or assembly within the endoplasmic reticulum (5).RIC-3 is a protein with two transmembrane domains followed by coiled-coil domains. When first identified, this protein showed no similarity to any characterized protein. Only one homolog was identified, the Drosophila CG9349 gene that is similar in both sequence and overall predicted structure. However, the demonstrated ability of RIC-3 to enhance whole-cell current amplitudes resulting from expression of both the C. elegans DEG-3/DES-2 and th...

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Members of the RIC-3 gene family are effectors of nicotinic acetylcholine receptor (nAChR) expression in vertebrates and invertebrates. In Caenorhabditis elegans RIC-3 is needed for functional expression of multiple nAChRs, including the DEG-3/DES-2 nAChR. Effects of RIC-3 on DEG-3/DES-2 functional expression are found in vivo and following heterologous expression in Xenopus leavis oocytes. We now show that in X. leavis oocytes RIC-3 also affects the kinetics and agonist affinity properties of the DEG-3/DES-2 receptor. Because these effects are mimicked by increasing the ratio of DEG-3 subunits within DEG-3/DES-2 receptors, this suggests that RIC-3 may preferentially promote maturation of DEG-3-rich receptors. Indeed, effects of RIC-3 on functional expression of DEG-3/DES-2 positively correlate with the DEG-3 to DES-2 ratio. All RIC-3 family members have two transmembrane domains followed by one or two coiled-coil domains. Here we show that the effects of RIC-3 on functional expression and on receptor properties are mediated by the transmembrane domains and do not require the coiled-coil domains. In agreement with this, mammals express a RIC-3 transcript lacking the coiled-coil domain that is capable of promoting DEG-3/DES-2 functional expression. Last, we show that RIC-3 affects DEG-3 quantity, suggesting stabilization of receptors or receptor intermediates by RIC-3. Together our results suggest that subunit-specific interactions of RIC-3 with nAChR subunits, mediated by the transmembrane domains, are sufficient for the effects of RIC-3 on nAChR quantity and quality.

Regulatory B cells have gained prominence in their role as modulators of the immune response against tumors, infectious diseases, and autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis, among others. The concept of regulatory B cells has been strongly associated with interleukin (IL)-10 production; however, there is growing evidence that supports the existence of other regulatory mechanisms, such as the production of transforming growth factor β (TGF-β), induced cell death of effector T cells, and the induction of CD4(+)CD25(-)Foxp3(+) regulatory T cells. The regulatory function of B cells has been associated with the presence and activation of molecules such as CD40, CD19, CD1d, and BCR. Alterations in signaling by any of these pathways leads to a marked defect in regulatory B cells and to increased clinical symptoms and proinflammatory signs, both in murine models and in autoimmune diseases in humans. B cells mainly exert their regulatory effect through the inhibition of proliferation and production of proinflammatory mediators, such as TNF-α, IFN-γ, and IL-17 by CD4(+) T cells. A better understanding of how regulatory B cells function will offer new perspectives with regard to the treatment of various human diseases.

Glycinergic inhibition plays a central role in the auditory brainstem circuitries involved in sound localization and in the encoding of temporal action potential firing patterns. Modulation of this inhibition has the potential to fine-tune information processing in these networks. Here we show that nitric oxide (NO) signaling in the auditory brainstem (where activity-dependent generation of NO is documented) modulates the strength of inhibition by changing the chloride equilibrium potential. Recent evidence demonstrates that large inhibitory postsynaptic currents (IPSCs) in neurons of the superior paraolivary nucleus (SPN) are enhanced by a very low intracellular chloride concentration, generated by the neuronal potassium chloride co-transporter (KCC2) expressed in the postsynaptic neurons. Our data show that modulation by NO caused a 15 mV depolarizing shift of the IPSC reversal potential, reducing the strength of inhibition in SPN neurons, without changing the threshold for action potential firing. Regulating inhibitory strength, through cGMP-dependent changes in the efficacy of KCC2 in the target neuron provides a postsynaptic mechanism for rapidly controlling the inhibitory drive, without altering the timing or pattern of the afferent spike train. Therefore, this NO-mediated suppression of KCC2 can modulate inhibition in one target nucleus (SPN), without influencing inhibitory strength of other target nuclei (MSO, LSO) even though they are each receiving collaterals from the same afferent nucleus (a projection from the medial nucleus of the trapezoid body, MNTB).

Dendritic cells (DCs) are critical in asthma and many other immune diseases. We previously demonstrated a role for PARP-1 in asthma. Evidence on PARP-1 playing a role in Th2-associated DC function is not clear. In this study, we examined whether PARP-1 is critical for DC differentiation and function using bone marrow progenitors and their migration to the lung in an ovalbumin-based mouse model of asthma. Results show that changes in PARP-1 levels during GM-CSF-induced DC differentiation from bone marrow progenitors were cyclic and appear to be part of an array of changes that included STAT3/STAT5/STAT6/GRAIL/RAD51. Interestingly, PARP-1 gene deletion affected primarily STAT6 and γH2AX. PARP-1 inhibition significantly reduced the migration of DCs to the lungs of ovalbumin-challenged mice, which was associated with a concomitant reduction in lung levels of the adhesion molecule VCAM-1. The requirement of PARP-1 for VCAM-1 expression was confirmed using endothelial and lung smooth muscle cells. PARP-1 expression and activity were also required for VCAM-1 in differentiated DCs. An assessment of CD11b+/CD11c+/MHCIIhigh DCs in spleens and lymph nodes of OVA-sensitized mice revealed that PARP-1 inhibition genetically or by olaparib exerted little to no effect on DC differentiation, percentage of CD80+/CD86+/CD40+-expressing cells, or their capacity to promote proliferation of ovalbumin-primed (OTII) CD4+ T cells. These findings were corroborated using GM-CSF-induced differentiation of DCs from the bone marrow. Surprisingly, the PARP-1−/− DCs exhibited a higher intrinsic capacity to induce OTII CD4+ T cell proliferation in the absence of ovalbumin. Overall, our results show that PARP-1 plays little to no role in DC differentiation and function and that the protective effect of PARP-1 inhibition against asthma is associated with a prevention of DC migration to the lung through a reduction in VCAM-1 expression. Given the current use of PARP inhibitors (e.g., olaparib) in the clinic, the present results may be of interest for the relevant therapies.