Real-time imaging of axonal membrane protein life cycles.

The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

Over-The-Counter Hearing Aids: Are They Safe and Effective?

OBJECTIVE: In 2022, the Food and Drug Administration established a new regulatory category for over-the-counter (OTC) hearing aids for mild to moderate hearing loss. Herein, we aim to better compare the safety and efficacy of these devices to that of prescription hearing aids. STUDY DESIGN: Comparative-effectiveness model. SETTING: Academic Audiology Center. METHODS: The safety and efficacy of prescription and OTC hearing aids was compared using the AudioScan Verifit 2 Testbox software. Three types of hearing loss (downsloping, sharp downsloping, and reverse sloping) were analyzed. Efficacy was tested at 3 volume inputs and was measured by calculating the average difference in test points (produced by the devices) and target points (estimated by the software). Safety was assessed by calculating the average difference in test points and the maximally safe hearing level (produced by the software). RESULTS: Prescription hearing aids were found to have a better safety profile by being further from the safety threshold compared to OTC devices at the 8000 Hz frequency for the 2 types of downsloping hearing loss patterns studied (48 vs 30.5 dB, P = .04; 51 vs 32.5 dB, P = .03). Prescription hearing aids also carried a statistically significant advantage at 3 test points. OTC hearing aids generally had a greater difference between test and target points. CONCLUSION: OTC and prescription hearing aids are comparably safe, though OTC hearing aids are slightly less efficacious. Further evaluation of the OTC hearing aid efficacy is warranted to ensure it provides the gain of benefit needed for different types of hearing loss.

Functionally-selective inhibition of threshold sodium currents and excitability in dorsal root ganglion neurons by cannabinol.

Cannabinol (CBN), an incompletely understood metabolite for ∆9-tetrahydrocannabinol, has been suggested as an analgesic. CBN interacts with endocannabinoid (CB) receptors, but is also reported to interact with non-CB targets, including various ion channels. We assessed CBN effects on voltage-dependent sodium (Nav) channels expressed heterologously and in native dorsal root ganglion (DRG) neurons. Our results indicate that CBN is a functionally-selective, but structurally-non-selective Nav current inhibitor. CBN's main effect is on slow inactivation. CBN slows recovery from slow-inactivated states, and hyperpolarizes steady-state inactivation, as channels enter deeper and slower inactivated states. Multielectrode array recordings indicate that CBN attenuates DRG neuron excitability. Voltage- and current-clamp analysis of freshly isolated DRG neurons via our automated patch-clamp platform confirmed these findings. The inhibitory effects of CBN on Nav currents and on DRG neuron excitability add a new dimension to its actions and suggest that this cannabinoid may be useful for neuropathic pain.

Compartment-specific regulation of NaV1.7 in sensory neurons after acute exposure to TNF-α.

Tumor necrosis factor α (TNF-α) is a major pro-inflammatory cytokine, important in many diseases, that sensitizes nociceptors through its action on a variety of ion channels, including voltage-gated sodium (NaV) channels. We show here that TNF-α acutely upregulates sensory neuron excitability and current density of threshold channel NaV1.7. Using electrophysiological recordings and live imaging, we demonstrate that this effect on NaV1.7 is mediated by p38 MAPK and identify serine 110 in the channel's N terminus as the phospho-acceptor site, which triggers NaV1.7 channel insertion into the somatic membrane. We also show that the N terminus of NaV1.7 is sufficient to mediate this effect. Although acute TNF-α treatment increases NaV1.7-carrying vesicle accumulation at axonal endings, we did not observe increased channel insertion into the axonal membrane. These results identify molecular determinants of TNF-α-mediated regulation of NaV1.7 in sensory neurons and demonstrate compartment-specific effects of TNF-α on channel insertion in the neuronal plasma membrane.

Sodium currents in naïve mouse dorsal root ganglion neurons: No major differences between sexes.

Sexual dimorphism has been reported in multiple pre-clinical and clinical studies on pain. Previous investigations have suggested that in at least some states, rodent dorsal root ganglion (DRG) neurons display differential sex-dependent regulation and expression patterns of various proteins involved in the pain pathway. Our goal in this study was to determine whether sexual dimorphism in the biophysical properties of voltage-gated sodium (Nav) currents contributes to these observations in rodents. We recently developed a novel method that enables high-throughput, unbiased, and automated functional analysis of native rodent sensory neurons from naïve WT mice profiled simultaneously under uniform experimental conditions. In our previous study, we performed all experiments in neurons that were obtained from mixed populations of adult males or females, which were combined into single (combined male/female) data sets. Here, we have re-analyzed the same previously published data and segregated the cells based on sex. Although the number of cells in our previously published data sets were uneven for some comparisons, our results do not show sex-dependent differences in the biophysical properties of Nav currents in these native DRG neurons.

The Posterior Trans-Septal Portal in Knee Arthroscopy Review of the Technique Evolution, Indications, and Technical Pearls.

Complete arthroscopic visualization of the posterior com-partment of the knee is limited when using the traditional anterior portals. The trans-septal portal technique, created in 1997, has allowed surgeons to view the complete posterior compartment of the knee in a less-invasive way compared to open surgery. Since the description of the posterior trans-septal portal, several authors have modified the technique. However, the paucity of literature describing the trans-septal portal technique hints that widespread arthroscopic adop-tion has not yet been achieved. While still in its infancy, the literature has cumulatively reported over 700 successful knee surgeries using the posterior trans-septal portal technique with no reports of neurovascular injury. However, creation of the trans-septal portal carries risks due to its close prox-imity to the popliteal and middle geniculate artery, giving surgeons little room for technical error when developing this portal. Knowledge of the posterior anatomy, the evolution of the trans-septal portal, and current recommendations and safety options for using the technique will benefit orthopedic surgeons looking to incorporate this technique into their surgical arsenal. Furthermore, utilization of the trans-septal portal technique offers a significant benefit to the surgical treatment of conditions that involve the need for posterior knee access or visualization.

Conserved but not critical: Trafficking and function of NaV1.7 are independent of highly conserved polybasic motifs.

Non-addictive treatment of chronic pain represents a major unmet clinical need. Peripheral voltage-gated sodium (NaV) channels are an attractive target for pain therapy because they initiate and propagate action potentials in primary afferents that detect and transduce noxious stimuli. NaV1.7 sets the gain on peripheral pain-signaling neurons and is the best validated peripheral ion channel involved in human pain, and previous work has shown that it is transported in vesicles in sensory axons which also carry Rab6a, a small GTPase known to be involved in vesicular packaging and axonal transport. Understanding the mechanism of the association between Rab6a and NaV1.7 could inform therapeutic modalities to decrease trafficking of NaV1.7 to the distal axonal membrane. Polybasic motifs (PBM) have been shown to regulate Rab-protein interactions in a variety of contexts. In this study, we explored whether two PBMs in the cytoplasmic loop that joins domains I and II of human NaV1.7 were responsible for association with Rab6a and regulate axonal trafficking of the channel. Using site-directed mutagenesis we generated NaV1.7 constructs with alanine substitutions in the two PBMs. Voltage-clamp recordings showed that the constructs retain wild-type like gating properties. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live sensory axons shows that mutations of these PBMs do not affect co-trafficking of Rab6a and NaV1.7, or the accumulation of the channel at the distal axonal surface. Thus, these polybasic motifs are not required for interaction of NaV1.7 with the Rab6a GTPase, or for trafficking of the channel to the plasma membrane.

Inflammation differentially controls transport of depolarizing Nav versus hyperpolarizing Kv channels to drive rat nociceptor activity.

Inflammation causes pain by shifting the balance of ionic currents in nociceptors toward depolarization, leading to hyperexcitability. The ensemble of ion channels within the plasma membrane is regulated by processes including biogenesis, transport, and degradation. Thus, alterations in ion channel trafficking may influence excitability. Sodium channel NaV1.7 and potassium channel KV7.2 promote and oppose excitability in nociceptors, respectively. We used live-cell imaging to investigate mechanisms by which inflammatory mediators (IM) modulate the abundance of these channels at axonal surfaces through transcription, vesicular loading, axonal transport, exocytosis, and endocytosis. Inflammatory mediators induced a NaV1.7-dependent increase in activity in distal axons. Further, inflammation increased the abundance of NaV1.7, but not of KV7.2, at axonal surfaces by selectively increasing channel loading into anterograde transport vesicles and insertion at the membrane, without affecting retrograde transport. These results uncover a cell biological mechanism for inflammatory pain and suggest NaV1.7 trafficking as a potential therapeutic target.

Paclitaxel effects on axonal localization and vesicular trafficking of NaV1.8.

Patients treated with paclitaxel (PTX) or other antineoplastic agents can experience chemotherapy-induced peripheral neuropathy (CIPN), a debilitating side effect characterized by numbness and pain. PTX interferes with microtubule-based transport, which inhibits tumor growth via cell cycle arrest but can also affect other cellular functions including trafficking of ion channels critical to transduction of stimuli by sensory neurons of the dorsal root ganglia (DRG). We examined the effects of PTX on voltage-gated sodium channel NaV1.8, which is preferentially expressed in DRG neurons, using a microfluidic chamber culture system and chemigenetic labeling to observe anterograde channel transport to the endings of DRG axons in real time. PTX treatment increased the numbers of NaV1.8-containing vesicles traversing the axons. Vesicles in PTX-treated cells exhibited greater average velocity, along with shorter and less frequent pauses along their trajectories. These events were paralleled by greater surface accumulation of NaV1.8 channels at the distal ends of DRG axons. These results were consistent with observations that NaV1.8 is trafficked in the same vesicles containing NaV1.7 channels, which are also involved in pain syndromes in humans and are similarly affected by PTX treatment. However, unlike Nav1.7, we did not detect increased NaV1.8 current density measured at the neuronal soma, suggesting a differential effect of PTX on trafficking of NaV1.8 in soma versus axonal compartments. Therapeutic targeting of axonal vesicular traffic would affect both Nav1.7 and Nav1.8 channels and increase the possibilities of alleviating pain associated with CIPN.

High-throughput combined voltage-clamp/current-clamp analysis of freshly isolated neurons.

The patch-clamp technique is the gold-standard methodology for analysis of excitable cells. However, throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for applications like drug screening, has been primarily used to study channels and receptors expressed in heterologous systems. We introduce an approach for automated high-throughput patch-clamping that enhances analysis of excitable cells at the channel and cellular levels. This involves dissociating and isolating neurons from intact tissues and patch-clamping using a robotic instrument, followed by using an open-source Python script for analysis and filtration. As a proof of concept, we apply this approach to investigate the biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables voltage- and current-clamp recordings in the same cell, allowing unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of freshly isolated neurons without requiring culturing on coverslips.

The fates of internalized NaV1.7 channels in sensory neurons: Retrograde cotransport with other ion channels, axon-specific recycling, and degradation.

Neuronal function relies on the maintenance of appropriate levels of various ion channels at the cell membrane, which is accomplished by balancing secretory, degradative, and recycling pathways. Neuronal function further depends on membrane specialization through polarized distribution of specific proteins to distinct neuronal compartments such as axons. Voltage-gated sodium channel NaV1.7, a threshold channel for firing action potentials in nociceptors, plays a major role in human pain, and its abundance in the plasma membrane is tightly regulated. We have recently characterized the anterograde axonal trafficking of NaV1.7 channels in Rab6A-positive vesicles, but the fate of internalized channels is not known. Membrane proteins that have undergone endocytosis can be directed into multiple pathways including those for degradation, recycling to the membrane, and transcytosis. Here, we demonstrate NaV1.7 endocytosis and dynein-dependent retrograde trafficking in Rab7-containing late endosomes together with other axonal membrane proteins using real-time imaging of live neurons. We show that some internalized NaV1.7 channels are delivered to lysosomes within the cell body, and that there is no evidence for NaV1.7 transcytosis. In addition, we show that NaV1.7 is recycled specifically to the axonal membrane as opposed to the soma membrane, suggesting a novel mechanism for the development of neuronal polarity. Together, these results shed light on the mechanisms by which neurons maintain excitable membranes and may inform efforts to target ion channel trafficking for the treatment of disorders of excitability.

Complex effects on CaV2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder.

P/Q-type Ca2+ currents mediated by CaV2.1 channels are essential for active neurotransmitter release at neuromuscular junctions and many central synapses. Mutations in CACNA1A, the gene encoding the principal CaV2.1 α1A subunit, cause a broad spectrum of neurological disorders. Typically, gain-of-function (GOF) mutations are associated with migraine and epilepsy while loss-of-function (LOF) mutations are causative for episodic and congenital ataxias. However, a cluster of severe CaV2.1 channelopathies have overlapping presentations which suggests that channel dysfunction in these disorders cannot always be defined bimodally as GOF or LOF. In particular, the R1667P mutation causes focal seizures, generalized hypotonia, dysarthria, congenital ataxia and, in one case, cerebral edema leading ultimately to death. Here, we demonstrate that the R1667P mutation causes both channel GOF (hyperpolarizing voltage-dependence of activation, slowed deactivation) and LOF (slowed activation kinetics) when expressed heterologously in tsA-201 cells. We also observed a substantial reduction in Ca2+ current density in this heterologous system. These changes in channel gating and availability/expression manifested in diminished Ca2+ flux during action potential-like stimuli. However, the integrated Ca2+ fluxes were no different when normalized to tail current amplitude measured upon repolarization from the reversal potential. In summary, our findings indicate a complex functional effect of R1667P and support the idea that pathological missense mutations in CaV2.1 may not represent exclusively GOF or LOF.

Depolarizing NaV and hyperpolarizing KV channels are co-trafficked in sensory neurons.

Neuronal excitability relies on coordinated action of functionally distinct ion channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance (OPAL) imaging to investigate trafficking of NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons - NaV1.6, NaV1.7, NaV1.8, and NaV1.9 - are co-transported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions - NaV1.7, KV7.2, and TNFR1 - are co-transported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein - NCX2 - is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.Significance StatementNormal neuronal excitability is dependent on precise regulation of membrane proteins including NaV and KV channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.

A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in CaV2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in CaV2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat CaV2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an ∼25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of CaV2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type CaV2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in CaV2.1.

Zebrafish as a Model System for the Study of Severe CaV2.1 (α1A) Channelopathies.

The P/Q-type CaV2.1 channel regulates neurotransmitter release at neuromuscular junctions (NMJ) and many central synapses. CACNA1A encodes the pore-containing α1A subunit of CaV2.1 channels. In humans, de novo CACNA1A mutations result in a wide spectrum of neurological, neuromuscular, and movement disorders, such as familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA2), as well as a more recently discovered class of more severe disorders, which are characterized by ataxia, hypotonia, cerebellar atrophy, and cognitive/developmental delay. Heterologous expression of CaV2.1 channels has allowed for an understanding of the consequences of CACNA1A missense mutations on channel function. In contrast, a mechanistic understanding of how specific CACNA1A mutations lead in vivo to the resultant phenotypes is lacking. In this review, we present the zebrafish as a model to both study in vivo mechanisms of CACNA1A mutations that result in synaptic and behavioral defects and to screen for effective drug therapies to combat these and other CaV2.1 channelopathies.

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p > 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.