Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remains a key open question. Here, whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. We show that this synapse elimination is dependent on the microglial fractalkine receptor, CX3CR1, but not complement receptor 3, signaling. Further, mice deficient in the CX3CR1 ligand (CX3CL1) also have profound defects in synapse elimination. Single-cell RNAseq then revealed that Cx3cl1 is cortical neuron-derived and Adam10 , a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and microglia following whisker lesioning. Finally, inhibition of Adam10 phenocopies Cx3cr1 −/− and Cx3cl1 −/− synapse elimination defects. Together, these results identify novel neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.

Summary Background Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes. Results Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channel rhodopsin elicited physical withdrawal symptoms in both nicotine-naïve and chronic nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blocking IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the β4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naïve mice and activated non-somatostatin neurons in the IPN. Conclusions Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.

Increased anxiety is a predominant withdrawal symptom in abstinent smokers, yet the neuroanatomical and molecular bases underlying it are unclear. Here, we show that withdrawal-induced anxiety increases activity of neurons in the interpeduncular intermediate (IPI), a subregion of the interpeduncular nucleus (IPN). IPI activation during nicotine withdrawal was mediated by increased corticotropin releasing factor (CRF) receptor-1 expression and signaling, which modulated glutamatergic input from the medial habenula (MHb). Pharmacological blockade of IPN CRF1 receptors or optogenetic silencing of MHb input reduced IPI activation and alleviated withdrawal-induced anxiety; whereas IPN CRF infusion in mice increased anxiety. We identified a meso-interpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knock-down of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signaling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

N-type Ca 2؉ channels participate in acute activity-dependent processes such as regulation of Ca 2؉ -activated K ؉ channels and in more prolonged events such as gene transcription and long-term depression. A slow postsynaptic M1 muscarinic receptor-mediated modulation of N-type current in superior cervical ganglion neurons may be important in regulating these processes. This slow pathway inhibits N-type current by using a diffusible second messenger that has remained unidentified for more than a decade. Using wholecell patch-clamp techniques, which isolate the slow pathway, we found that the muscarinic agonist oxotremorine methiodide not only inhibits currents at positive potentials but enhances N-type current at negative potentials. Enhancement was also observed in cell-attached patches. These findings provide evidence for N-type Ca 2؉ -current enhancement by a classical neurotransmitter. Moreover, enhancement and inhibition of current by oxotremorine methiodide mimics modulation observed with direct application of a low concentration of arachidonic acid (AA). Although no transmitter has been reported to use AA as a second messenger to modulate any Ca 2؉ current in either neuronal or nonneuronal cells, we nevertheless tested whether a fatty acid signaling cascade was involved. Blocking phospholipase C, phospholipase A2, or AA but not AA metabolism minimized muscarinic modulation of N-type current, supporting the participation of these molecules in the slow pathway. A role for the G protein Gq was also confirmed by blocking muscarinic modulation of Ca 2؉ currents with anti-Gq␣ antibody. Our finding that AA participates in the slow pathway strongly suggests that it may be the previously unknown diffusible second messenger.bovine serum albumin ͉ calcium channel ͉ Gq protein ͉ M1 muscarinic receptor ͉ phospholipase A2 ͉

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MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.

We recently reported that arachidonic acid (AA) inhibits L- and N-type Ca(2+) currents at positive test potentials in the presence of the dihydropyridine L-type Ca(2+) channel agonist (+)-202-791 in dissociated neonatal rat superior cervical ganglion neurons [Liu L and Rittenhouse AR. J Physiol (Lond) 525: 291-404, 2000]. In this first of two companion papers, we characterized the mechanism of inhibition by AA at the whole cell level. In the presence of either omega-conotoxin GVIA or nimodipine, AA decreased current amplitude, confirming that L- and N-type currents, respectively, were inhibited. AA-induced inhibition was concentration dependent and reversible with an albumin-containing wash solution, but appears independent of AA metabolism and G protein activity. In characterizing inhibition, an AA-induced enhancement of current amplitude was revealed that occurred primarily at negative test potentials. Cell dialysis with albumin minimized inhibition but had little effect on enhancement, suggesting that AA has distinct sites of action. We examined AA's actions on current kinetics and found that AA increased holding potential-dependent inactivation. AA also enhanced the rate of N-type current activation. These findings indicate that AA causes multiple changes in sympathetic Ca(2+) currents.

Nicotine is reinforcing because it activates dopaminergic (DAergic) neurons within the ventral tegmental area (VTA) of the brain's mesocorticolimbic reward circuitry. This increase in activity can occur for a period of several minutes up to an hour and is thought to be a critical component of nicotine dependence. However, nicotine concentrations that are routinely self-administered by smokers are predicted to desensitize high-affinity ␣4␤2 neuronal nicotinic acetylcholine receptors (nAChRs) in seconds. Thus, how physiologically relevant nicotine concentrations persistently activate VTA DAergic neurons is unknown. Here we show that nicotine can directly and robustly increase the firing frequency of VTA DAergic neurons for several minutes. In mouse midbrain slices, 300 nM nicotine elicited a persistent inward current in VTA DAergic neurons that was blocked by ␣-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the ␣6 subunit. ␣-conotoxin MII[H9A;L15A] also significantly reduced the longlasting increase in DAergic neuronal activity produced by low concentrations of nicotine. In addition, nicotine failed to significantly activate VTA DAergic neurons in mice that did not express either ␣4 or ␣6 nAChR subunits. Conversely, selective activation of nAChRs containing the ␣4 subunit in knock-in mice expressing a hypersensitive version of these receptors yielded a biphasic response to nicotine consisting of an acute desensitizing increase in firing frequency followed by a sustained increase that lasted several minutes and was sensitive to ␣-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing ␣4 and ␣6 subunits.