Keith S. Elmslie

Small diameter unmyelinated (group IV) and myelinated (group III) muscle afferents transmit action potentials to signal muscle pain and the exercise pressor reflex (EPR). The EPR helps to drive the cardiovascular response to exercise. µ- (µOR), δ- (δOR), and κ - (κOR) opioid receptors (OR) have been shown to be expressed by dorsal root ganglia (DRG) neurons, but only peripheral µOR activation has been shown to inhibit the EPR mediated by group IV afferents. To determine if this results from differential OR expression, we identified group IV neurons in DRG and axons in gastrocnemius muscles from both male and female rats, and used immunocytochemistry to compare the expression of µOR, δOR, and κOR. We show that µOR labeling was more frequently observed in group IV somata and axons vs. δOR or κOR. OR axonal labeling was lower than somata labeling, particularly for δOR & κOR. There were no significant differences in OR staining between male and female animals. The relatively high µOR axonal expression suggests that µOR are selectively transported to the peripheral group IV axons and can explain the selective effect of µOR agonists to inhibit group IV afferent activity that generates the EPR and muscle pain.

G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that Na_V1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of Na_V1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

K_V7 channels control neuronal excitability. We show that these channels are expressed in muscle afferents and generate currents that are blocked by XE991 and bradykinin (BK). The XE991 block suggests that K_V7 current comprises K_V7.2/3 and K_V7.5 channels. The BK inhibition of K_V7 channels may explain how BK activates the exercise pressor reflex (EPR). Retigabine can enhance K_V7 current, which could help control the inappropriately activated EPR in patients with cardiovascular disease.

The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, Na_V1.6, and Na_V1.7) and -resistant (TTX-R, Na_V1.8, and Na_V1.9) voltage-gated sodium (Na_V) channels, but Na_V1.9 current has not yet been demonstrated. Using a F⁻-containing internal solution, we found a Na_Vcurrent in muscle afferent neurons that activates at around −70 mV with slow activation and inactivation kinetics, as expected from Na_V1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following Na_V1.9 current rundown (internal F⁻), application of the Na_V1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl⁻), which suggests that Na_V1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were Na_V1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also Na_V1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of Na_V1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR.NEW & NOTEWORTHY Small diameter muscle afferents signal pain and muscle activity levels. The muscle activity signals drive the cardiovascular system to increase muscle blood flow, but these signals can become exaggerated in cardiovascular disease to exacerbate cardiac damage. The voltage-dependent sodium channel Na_V1.9 plays a unique role in controlling afferent excitability. We show that Na_V1.9 channels are expressed in muscle afferents, which supports these channels as a target for drug development to control hyperactivity of these neurons.

Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules α-neurexins (αNrxns), the extracellularly longer variants of the three vertebrateNrxngenes. Ca²⁺influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (Ca_V2.1) triggers release of fusion-ready vesicles at many boutons. α2δ Auxiliary subunits regulate trafficking and kinetic properties of Ca_V2.1 pore-forming subunits but it has remained unclear if this involves αNrxns. Using live cell imaging with Ca²⁺indicators, we report here that the total presynaptic Ca²⁺influx in primary hippocampal neurons of αNrxn triple knock-out mice of both sexes is reduced and involved lower Ca_V2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of Ca_V2.1 pore-forming subunits, and elevated surface mobility of α2δ-1 on axons. Overexpression of Nrxn1α in αNrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca²⁺influx and synaptic vesicle release. Moreover, coexpression of Nrxn1α together with α2δ-1 subunits facilitates Ca²⁺influx further but causes little augmentation together with a different subunit, α2δ-3, suggesting remarkable specificity. Expression of defined recombinant Ca_V2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1α in combination with α2δ-1, but not with α2δ-3, facilitates Ca²⁺currents of recombinant Ca_V2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1α acts as a positive regulator of Ca²⁺influx through Ca_V2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENTSynaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their α2δ-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.

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 GABA_B 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 GABA_B 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. Significance This study addresses the need to phase out opioids as the major analgesic drugs for moderate to severe chronic pain. We establish that a highly selective and potent inhibitor of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype prevents the expression of chemotherapy-induced neuropathic pain. Thus, selective antagonists of the α9α10 nAChR are potential leads for nonopioid analgesic drug development. The effects of inhibitors of the α9α10 receptor, together with genetic studies, suggest a key role for the α9α10 nAChR subtype in an intercellular signaling network that can be activated by diverse insults (e.g., chemotherapy, nerve injury, and diabetes).

Patients with intermittent claudication suffer from both muscle pain and an exacerbated exercise pressor reflex. Excitability of the group III and group IV afferent fibers mediating these functions is controlled in part by voltage-dependent sodium (Na_V) channels. We previously found tetrodotoxin-resistant Na_V1.8 channels to be the primary type in muscle afferent somata. However, action potentials in group III and IV afferent axons are blocked by TTX, supporting a minimal role of Na_V1.8 channels. To address these apparent differences in Na_V channel expression between axon and soma, we used immunohistochemistry to identify the Na_V channels expressed in group IV axons within the gastrocnemius muscle and the dorsal root ganglia sections. Positive labeling by an antibody against the neurofilament protein peripherin was used to identify group IV neurons and axons. We show that >67% of group IV fibers express Na_V1.8, Na_V1.6, or Na_V1.7. Interestingly, expression of Na_V1.8 channels in group IV somata was significantly higher than in the fibers, whereas there were no significant differences for either Na_V1.6 or Na_V1.7. When combined with previous work, our results suggest that Na_V1.8 channels are expressed in most group IV axons, but that, under normal conditions, Na_V1.6 and/or Na_V1.7 play a more important role in action potential generation to signal muscle pain and the exercise pressor reflex.

Significance: The α9α10 nicotinic AChR (nAChR) subtype is a recently identified target for the development of breast cancer chemotherapeutics and analgesics, particularly to treat neuropathic pain. Structure/function analyses of antagonists of this subtype are therefore essential for the development of specific therapeutic compounds. TheConusgenus is a rich source of pharmacologically active peptides, and we report here that the αO-conotoxin GeXIVA is a potent and selective antagonist of the α9α10 nAChR subtype. GeXIVA displays unique structural properties among otherConuspeptides and represents a previously unidentified template for molecules active against neuropathic pain.

Chronic pain is very difficult to treat. Thus, novel analgesics are a critical area of research. Strong preclinical evidence supports the analgesic effects of α-conopeptides, Vc1.1 and RgIA, which block α9α10 nicotinic acetylcholine receptors (nAChRs). However, the analgesic mechanism is controversial. Some evidence supports the block of α9α10 nAChRs as an analgesic mechanism, while other evidence supports the inhibition of N-type Ca_V(Ca_V2.2) current via activation of GABA_Breceptors. Here, we reassess the effect of Vc1.1 and RgIA on Ca_Vcurrent in rat sensory neurons. Unlike the previous findings, we found highly variable effects among individual sensory neurons, but on average only minimal inhibition induced by Vc1.1, and no significant effect on the current by RgIA. We also investigated the potential involvement of GABA_Breceptors in the Vc1.1-induced inhibition, and found no correlation between the size of Ca_Vcurrent inhibition induced by baclofen (GABA_Bagonist) versus that induced by Vc1.1. Thus, GABA_Breceptors are unlikely to mediate the Vc1.1-induced Ca_Vcurrent inhibition. Based on the present findings, Ca_Vcurrent inhibition in dorsal root ganglia is unlikely to be the predominant mechanism by which either Vc1.1 or RgIA induce analgesia.