A major dose-limiting side effect associated with cancer-treating antineoplastic drugs is the development of neuropathic pain, which is not readily relieved by available analgesics. A better understanding of the mechanisms that underlie pain generation has potential to provide targets for prophylactic management of chemotherapy pain. Here, we delineate a pathway for pain that is induced by the chemotherapeutic drug vincristine sulfate (VCR). In a murine model of chemotherapy-induced allodynia, VCR treatment induced upregulation of endothelial cell adhesion properties, resulting in the infiltration of circulating CX3CR1 + monocytes into the sciatic nerve. At the endothelial-nerve interface, CX3CR1 + monocytes were activated by the chemokine CX3CL1 (also known as fractalkine [FKN]), which promoted production of reactive oxygen species that in turn activated the receptor TRPA1 in sensory neurons and evoked the pain response. Furthermore, mice lacking CX3CR1 exhibited a delay in the development of allodynia following VCR administration. Together, our data suggest that CX3CR1 antagonists and inhibition of FKN proteolytic shedding, possibly by targeting ADAM10/17 and/or cathepsin S, have potential as peripheral approaches for the prophylactic treatment of chemotherapy-induced pain.

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M 4 muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligandbinding studies revealed that LY2033298 displayed a preference for the active state of the M 4 mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [ 35 S]GTPgS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3b phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M 4 mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [ 3 H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M 4 mAChR knockout mice, validating the M 4 mAChR as a key target of action of this novel allosteric ligand.

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Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca 2+ -dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca 2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca 2+ signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 lM) generated a rapid elevation in cytoplasmic Ca 2+ that was partially and additively inhibited (40%) by a7 and a3b2* nAChR subtype selective antagonists; a3b4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl 2 (100 lM) indicates that voltage-operated Ca 2+ channels (VOCC) mediate most of the nicotine-evoked response, although the a7 selective antagonist a-bungarotoxin (40 nM) further decreased the CdCl 2 -resistant component. The elevation of intracellular Ca 2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca 2+ stores were implicated in both the initial and sustained nicotine-evoked Ca 2+ responses, by the blockade observed after ryanodine (30 lM) and the inositoltriphosphate (IP 3 )-receptor antagonist, xestospongin-c (10 lM). Thus, nAChR subtypes are differentially coupled to specific sources of Ca 2+ : activation of nAChR induces a sustained elevation of intracellular Ca 2+ levels which is highly dependent on the activation of VOCC, and also involves Ca 2+ release from ryanodine and IP 3 -dependent intracellular stores. Moreover, the a7, but not a3b2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca 2+ increase that appears to be functionally coupled to ryanodine sensitive Ca 2+ stores.

Sazetidine-A has been recently proposed to be a "silent desensitizer" of ␣4␤2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes ␣4␤2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of ␣4␤2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by ␣4␤2* and ␣6␤2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant ␣4␤2 nAChRs in more detail. We expressed the two alternative stoichiometries of ␣4␤2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both ␣4(2)␤2(3)and ␣4(3)␤2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of ␣4␤2 nAChR: it was a full agonist on ␣4(2)␤2(3) nAChRs, whereas it had an efficacy of only 6% on ␣4(3)␤2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant ␣4␤2 nAChRs but shows differential efficacy on ␣4␤2 nAChRs subtypes.

The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

Substitution to the nicotine discriminative stimulus required high-affinity and high intrinsic activity at beta2 but not at beta4- or at alpha7-containing nicotinic receptors.

The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the ␣7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [ 3 H]dopamine release from rat striatal synaptosomes stimulated with 10 M nicotine. Other ␣7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [ 3 H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 M) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to ␣-conotoxin-MII (␣-CTx-MII). MLA was not additive with a maximally effective concentration of ␣-CTx-MII (100 nM) in inhibiting [3 H]dopamine release elicited by 10 M nicotine or 0.2 M UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with 125 I-␣-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens with B max values of 9.8 and 16.5 fmol/mg of protein, and K d values of 0.63 and 0.83 nM, respectively. MLA fully inhibited 125 I-␣-CTx-MII binding to striatum and nucleus accumbens with a K i value of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and ␣-CTx-MII interact with a presynaptic nAChR of subunit composition ␣3/␣6␤2␤3* on dopamine neurons. The use of MLA as an ␣7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.