Conotoxins Targeting Voltage-Gated Sodium Ion Channels.

Voltage-gated sodium (NaV) channels are intimately involved in the generation and transmission of action potentials, and dysfunction of these channels may contribute to nervous system diseases, such as epilepsy, neuropathic pain, psychosis, autism, and cardiac arrhythmia. Many venom peptides selectively act on NaV channels. These include conotoxins, which are neurotoxins secreted by cone snails for prey capture or self-defense but which are also valuable pharmacological tools for the identification and/or treatment of human diseases. Typically, conotoxins contain two or three disulfide bonds, and these internal crossbraces contribute to conotoxins having compact, well defined structures and high stability. Of the conotoxins containing three disulfide bonds, some selectively target mammalian NaV channels and can block, stimulate, or modulate these channels. Such conotoxins have great potential to serve as pharmacological tools for studying the functions and characteristics of NaV channels or as drug leads for neurologic diseases related to NaV channels. Accordingly, discovering or designing conotoxins targeting NaV channels with high potency and selectivity is important. The amino acid sequences, disulfide bond connectivity, and three-dimensional structures are key factors that affect the biological activity of conotoxins, and targeted synthetic modifications of conotoxins can greatly improve their activity and selectivity. This review examines NaV channel-targeted conotoxins, focusing on their structures, activities, and designed modifications, with a view toward expanding their applications. SIGNIFICANCE STATEMENT: NaV channels are crucial in various neurologic diseases. Some conotoxins selectively target NaV channels, causing either blockade or activation, thus enabling their use as pharmacological tools for studying the channels' characteristics and functions. Conotoxins also have promising potential to be developed as drug leads. The disulfide bonds in these peptides are important for stabilizing their structures, thus leading to enhanced specificity and potency. Together, conotoxins targeting NaV channels have both immediate research value and promising future application prospects.

Analogs of α-conotoxin PnIC selectively inhibit α7ß2- over α7-only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.

Computational Design of α-Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.

Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

αO-Conotoxin GeXIVA[1,2] Reduced Neuropathic Pain and Changed Gene Expression in Chronic Oxaliplatin-Induced Neuropathy Mice Model.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting painful neuropathy that occurs commonly during cancer management, which often leads to the discontinuation of medication. Previous studies suggest that the α9α10 nicotinic acetylcholine receptor (nAChR)-specific antagonist αO-conotoxin GeXIVA[1,2] is effective in CIPN models; however, the related mechanisms remain unclear. Here, we analyzed the preventive effect of GeXIVA[1,2] on neuropathic pain in the long-term oxaliplatin injection-induced CIPN model. At the end of treatment, lumbar (L4-L6) spinal cord was extracted, and RNA sequencing and bioinformatic analysis were performed to investigate the potential genes and pathways related to CIPN and GeXIVA[1,2]. GeXIVA[1,2] inhibited the development of mechanical allodynia induced by chronic oxaliplatin treatment. Repeated injections of GeXIVA[1,2] for 3 weeks had no effect on the mice's normal pain threshold or locomotor activity and anxiety-like behavior, as evaluated in the open field test (OFT) and elevated plus maze (EPM). Our RNA sequencing results identified 209 differentially expressed genes (DEGs) in the CIPN model, and simultaneously injecting GeXIVA[1,2] with oxaliplatin altered 53 of the identified DEGs. These reverted genes were significantly enriched in immune-related pathways represented by the cytokine-cytokine receptor interaction pathway. Our findings suggest that GeXIVA[1,2] could be a potential therapeutic compound for chronic oxaliplatin-induced CIPN management.

Using Constellation Pharmacology to Characterize a Novel α-Conotoxin from Conus ateralbus.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3ß4, α6/α3ß4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3ß4 and α6/α3ß4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

Cross Talk between α7 and α3ß4 Nicotinic Receptors Prevents Their Desensitization in Human Chromaffin Cells.

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3ß4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3ß4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3ß4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3ß4 subtype is dependent on Ca2+ In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+ In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3ß4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3ß4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+ to cooperate in the case of the α3ß4 current increase. Because choline is an α3ß4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.

Differential tyrosine and serine/threonine phosphorylation/dephosphorylation pathways regulate the expression of α7 versus α3ß4 nicotinic receptor subtypes in mouse hippocampal neurons.

We have recently reported that α7 and α3ß4 nicotinic acetylcholine receptor (nAChR) subtypes are expressed in human chromaffin cells in the plasma membrane where they colocalize and physically interact. The present study was designed to evaluate whether those receptor subtypes also colocalize at the central nervous system to mutually interact, and whether their expression and colocalization are regulated by phosphorylation/dephosphorylation processes, as they are in human chromaffin cells. We have here found that in isolated and maintained in culture mouse hippocampal neurons, nAChR expression and colocalization of α7, but not α3ß4, nAChR subtypes decreased by tyrosine (Tyr)- and serine/threonine (Ser/Thr)-phosphatase inhibition. However, Tyr-kinase inhibition or protein-phosphatase 2A (PP2A) activation increased α3ß4 nAChR expression, diminishing receptor subtypes colocalization. Furthermore, colocalization is not recovered if the inhibitors of Tyr-phosphatase and kinases, or the inhibitor of Ser/Thr-phosphatases and the activator of PP2A are applied together. Therefore, regulation of α7 and α3ß4 nAChR subtypes expression by Tyr- and Ser/Thr kinases and phosphatases exhibit differential mechanisms in mouse hippocampal neurons. Colocalization of nAChR subtypes, however, is altered by any maneuver that affects these kinases or phosphatases, which might have consequences in the functional activity of nAChR subtypes.

Nicotinic acetylcholine receptors: Therapeutic targets for novel ligands to treat pain and inflammation.

Nicotinic acetylcholine receptors (nAChRs) have been historically defined as ligand-gated ion channels and function as such in the central and peripheral nervous systems. Recently, however, non-ionic signaling mechanisms via nAChRs have been demonstrated in immune cells. Furthermore, the signaling pathways where nAChRs are expressed can be activated by endogenous ligands other than the canonical agonists acetylcholine and choline. In this review, we discuss the involvement of a subset of nAChRs containing α7, α9, and/or α10 subunits in the modulation of pain and inflammation via the cholinergic anti-inflammatory pathway. Additionally, we review the most recent advances in the development of novel ligands and their potential as therapeutics.

Conus regius-Derived Conotoxins: Novel Therapeutic Opportunities from a Marine Organism.

Conus regius is a marine venomous mollusk of the Conus genus that captures its prey by injecting a rich cocktail of bioactive disulfide bond rich peptides called conotoxins. These peptides selectively target a broad range of ion channels, membrane receptors, transporters, and enzymes, making them valuable pharmacological tools and potential drug leads. C. regius-derived conotoxins are particularly attractive due to their marked potency and selectivity against specific nicotinic acetylcholine receptor subtypes, whose signalling is involved in pain, cognitive disorders, drug addiction, and cancer. However, the species-specific differences in sensitivity and the low stability and bioavailability of these conotoxins limit their clinical development as novel therapeutic agents for these disorders. Here, we give an overview of the main pharmacological features of the C. regius-derived conotoxins described so far, focusing on the molecular mechanisms underlying their potential therapeutic effects. Additionally, we describe adoptable chemical engineering solutions to improve their pharmacological properties for future potential clinical translation.

Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Efferent synaptic transmission at the vestibular type II hair cell synapse.

In the vestibular peripheral organs, type I and type II hair cells (HCs) transmit incoming signals via glutamatergic quantal transmission onto afferent nerve fibers. Additionally, type I HCs transmit via "non-quantal" transmission to calyx afferent fibers, by accumulation of glutamate and potassium in the synaptic cleft. Vestibular efferent inputs originating in the brainstem contact type II HCs and vestibular afferents. Here, synaptic inputs to type II HCs were characterized by using electrical and optogenetic stimulation of efferent fibers combined with in vitro whole cell patch-clamp recording from type II HCs in the rodent vestibular crista. Properties of efferent synaptic currents in type II HCs were similar to those found in cochlear HCs and mediated by activation of α9-containing nicotinic acetylcholine receptors (nAChRs) and small-conductance calcium-activated potassium (SK) channels. While efferents showed a low probability of release at low frequencies of stimulation, repetitive stimulation resulted in facilitation and increased probability of release. Notably, the membrane potential of type II HCs during optogenetic stimulation of efferents showed a strong hyperpolarization in response to single pulses and was further enhanced by repetitive stimulation. Such efferent-mediated inhibition of type II HCs can provide a mechanism to adjust the contribution of signals from type I and type II HCs to vestibular nerve fibers, with a shift of the response to be more like that of calyx-only afferents with faster non-quantal responses.NEW & NOTEWORTHY Type II vestibular hair cells (HCs) receive inputs from efferent neurons in the brain stem. We used in vitro optogenetic and electrical stimulation of vestibular efferent fibers to study their synaptic inputs to type II HCs. Stimulation of efferents inhibited type II HCs, similar to efferent effects on cochlear HCs. We propose that efferent inputs adjust the contribution of signals from type I and II HCs to vestibular nerve fibers.

Expression of α3ß2ß4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists.

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, ß2, and ß4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3ß2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3ß4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3ß4 nAChRs alone or α3ß4 together with the α3ß2ß4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3ß2ß4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.

Neuronal nicotinic acetylcholine receptors mediate ∆9 -THC dependence: Mouse and human studies.

Cessation from prolonged use of ∆9 -tetrahydrocannabinol (THC), the primary active compound responsible for the cannabimimetic effects of cannabis, results in a mild to moderate withdrawal syndrome in humans and laboratory animals. Whereas manipulations of the endogenous cannabinoid system (eg, cannabinoid receptors and endocannabinoid regulating enzymes) alter nicotine withdrawal, in this study we asked the reciprocal question. Do nicotinic acetylcholine receptors (nAChRs) modulate THC withdrawal? To assess the role of different nAChR subtypes in THC withdrawal, we used transgenic mouse, preclinical pharmacological, and human genetic correlation approaches. Our findings show that selective α3ß4* nAChR antagonist, AuIB, and α3ß4* nAChR partial agonist, AT-1001, dose-dependently attenuated somatic withdrawal signs in THC-dependent mice that were challenged with the cannabinoid-1 receptor antagonist rimonabant. Additionally, THC-dependent α5 and α6 nAChR knockout (KO) mice displayed decreased rimonabant precipitated somatic withdrawal signs compared with their wild-type counterparts. In contrast, ß2 and α7 nAChR KO mice showed no alterations in THC withdrawal signs. Moreover, deletion of ß2 nAChR did not alter the reduced expression of somatic signs by the preferred α6ß4* antagonist, BulA [T5A;P60]. Finally, the human genetic association studies indicated that variations in the genes that code for the α5, α3, ß4, and α6 nAChRs were associated with cannabis disorder phenotypes. Overall, these findings suggest that α3ß4* and α6ß4* nAChR subtypes represent viable targets for the development of medications to counteract THC dependence.

Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain.

Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.

Muscle Nicotinic Acetylcholine Receptors May Mediate Trans-Synaptic Signaling at the Mouse Neuromuscular Junction.

Block of neurotransmitter receptors at the neuromuscular junction (NMJ) has been shown to trigger upregulation of the number of synaptic vesicles released (quantal content, QC), a response termed homeostatic synaptic plasticity. The mechanism underlying this plasticity is not known. Here, we used selective toxins to demonstrate that block of α1-containing nicotinic acetylcholine receptors (nAChRs) at the NMJ of male and female mice triggers the upregulation of QC. Reduction of current flow through nAChRs, induced by drugs with antagonist activity, demonstrated that reduction in synaptic current per se does not trigger upregulation of QC. These data led to the remarkable conclusion that disruption of synaptic transmission is not sensed to trigger upregulation of QC. During studies of the effect of partial block of nAChRs on QC, we observed a small but reproducible increase in the decay kinetics of miniature synaptic currents. The change in kinetics was correlated with the increase in QC and raises the possibility that a change in postsynaptic nAChR conformation may be associated with the presynaptic increase in QC. We propose that, in addition to functioning in synaptic transmission, ionotropic muscle nicotonic nAChRs may serve as signaling molecules that participate in synaptic plasticity. Because nAChRs have been implicated in a number of disease states, the finding that nAChRs may be involved in triggering synaptic plasticity could have wide-reaching implications.SIGNIFICANCE STATEMENT The signals that initiate synaptic plasticity of the nervous system are still incompletely understood. Using the mouse neuromuscular junction as a model synapse, we studied how block of neurotransmitter receptors is sensed to trigger synaptic plasticity. Our studies led to the surprising conclusion that neither changes in synaptic current nor spiking of the presynaptic or postsynaptic cell are sensed to initiate synaptic plasticity. Instead, postsynaptic nicotinic acetylcholine receptors (nAChRs), in addition to functioning in synaptic transmission, may serve as signaling molecules that trigger synaptic plasticity. Because nAChRs have been implicated in a number of disease states, the finding that they may mediate synaptic plasticity has broad implications.

Molecular determinants of α-conotoxin potency for inhibition of human and rat α6ß4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and ß4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat α6/α3ß4 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to α-conotoxins. Sequence homology comparisons of human and rat α6ß4 nAChR subunits indicated that α6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of ß4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of α-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro13 in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu119 of human ß4 contributes to higher sensitivity of human α6/α3ß4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat α6/α3ß4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 and residue 119 of the ß4 subunit. These results highlight the potential significance of species differences in α6ß4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.

Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission.

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse ß2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.

RgIA4 Accelerates Recovery from Paclitaxel-Induced Neuropathic Pain in Rats.

Chemotherapeutic drugs are widely utilized in the treatment of human cancers. Painful chemotherapy-induced neuropathy is a common, debilitating, and dose-limiting side effect for which there is currently no effective treatment. Previous studies have demonstrated the potential utility of peptides from the marine snail from the genus Conus for the treatment of neuropathic pain. α-Conotoxin RgIA and a potent analog, RgIA4, have previously been shown to prevent the development of neuropathy resulting from the administration of oxaliplatin, a platinum-based antineoplastic drug. Here, we have examined its efficacy against paclitaxel, a chemotherapeutic drug that works by a mechanism of action distinct from that of oxaliplatin. Paclitaxel was administered at 2 mg/kg (intraperitoneally (IP)) every other day for a total of 8 mg/kg. Sprague Dawley rats that were co-administered RgIA4 at 80 µg/kg (subcutaneously (SC)) once daily, five times per week, for three weeks showed significant recovery from mechanical allodynia by day 31. Notably, the therapeutic effects reached significance 12 days after the last administration of RgIA4, which is suggestive of a rescue mechanism. These findings support the effects of RgIA4 in multiple chemotherapeutic models and the investigation of α9α10 nicotinic acetylcholine receptors (nAChRs) as a non-opioid target in the treatment of chronic pain.

Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain.

We have tested the hypothesis that neuropathic pain acting as a stressor drives functional plasticity in the sympathoadrenal system. The relation between neuropathic pain and adrenal medulla function was studied with behavioral, immunohistochemical and electrophysiological techniques in rats subjected to chronic constriction injury of the sciatic nerve. In slices of the adrenal gland from neuropathic animals, we have evidenced increased cholinergic innervation and spontaneous synaptic activity at the splanchnic nerve⁻chromaffin cell junction. Likewise, adrenomedullary chromaffin cells displayed enlarged acetylcholine-evoked currents with greater sensitivity to α-conotoxin RgIA, a selective blocker of α9 subunit-containing nicotinic acetylcholine receptors, as well as increased exocytosis triggered by voltage-activated Ca2+ entry. Altogether, these adaptations are expected to facilitate catecholamine output into the bloodstream. Last, but most intriguing, functional and immunohistochemical data indicate that P2X3 and P2X7 purinergic receptors and transient receptor potential vanilloid-1 (TRPV1) channels are overexpressed in chromaffin cells from neuropathic animals. These latter observations are reminiscent of molecular changes characteristic of peripheral sensitization of nociceptors following the lesion of a peripheral nerve, and suggest that similar phenomena can occur in other tissues, potentially contributing to behavioral manifestations of neuropathic pain.

Human nicotinic receptors in chromaffin cells: characterization and pharmacology.

During the last 10 years, we have been working on human chromaffin cells obtained from the adrenal gland of organ donors that suffered encephalic or cardiac death. We first electrophysiologically characterized the nicotinic acetylcholine receptors (nAChRs) activated by acetylcholine, and their contribution to the exocytosis of chromaffin vesicles and release of catecholamines. We have shown that these cells possess an adrenergic phenotype. This phenotype may contribute to an increased expression of α7 nAChRs in these cells, allowing for recording of α7 nAChR currents, something that had previously not been achieved in non-human species. The use of α-conotoxins allowed us to characterize non-α7 nAChR subtypes and, together with molecular biology experiments, conclude that the predominant nAChR subtype in human chromaffin cells is α3ß4* (asterisk indicates the posible presence of additional subunits). In addition, there is a minor population of αxß2 nAChRs. Both α7 and non-α7 nAChR subtypes contribute to the exocytotic process. Exocytosis mediated by nAChRs could be as large in magnitude as that elicited by calcium entry through voltage-dependent calcium channels. Finally, we have also investigated the effect of nAChR-targeted tobacco cessation drugs on catecholamine release in chromaffin cells. We have concluded that at therapeutic concentrations, varenicline alone does not increase the frequency of action potentials evoked by ACh. However, varenicline in the presence of nicotine does increase this frequency, and thus, in the presence of both drugs, the probability of increased catecholamine release in human chromaffin cells is high.