Thermal escape box: A cost-benefit evaluation paradigm for investigating thermosensation and thermal pain.

Thermosensation, the ability to detect and estimate temperature, is an evolutionarily conserved process that is essential for survival. Thermosensing is impaired in various pain syndromes, resulting in thermal allodynia, the perception of an innocuous temperature as painful, or thermal hyperalgesia, an exacerbated perception of a painful thermal stimulus. Several behavioral assays exist to study thermosensation and thermal pain in rodents, however, most rely on reflexive withdrawal responses or the subjective quantification of spontaneous nocifensive behaviors. Here, we created a new apparatus, the thermal escape box, which can be attached to temperature-controlled plates and used to assess temperature-dependent effort-based decision-making. The apparatus consists of a light chamber with an opening that fits around temperature-controlled plates, and a small entryway into a dark chamber. A mouse must choose to stay in a brightly lit aversive area or traverse the plates to escape to the enclosed dark chamber. We quantified escape latencies of adult C57Bl/6 mice at different plate temperatures from video recordings and found they were significantly longer at 5 °C, 18 °C, and 52 °C, compared to 30 °C, a mouse's preferred ambient temperature. Differences in escape latencies were abolished in male Trpm8-/- mice and in male Trpv1-/- animals. Finally, we show that chronic constriction injury procedures or oxaliplatin treatement significantly increased escape latencies at cold temperatures compared to controls, the later of which was prevented by the analgesic meloxicam. This demonstrates the utility of this assay in detecting cold pain. Collectively, our study has identified a new and effective tool that uses cost-benefit valuations to study thermosensation and thermal pain.

SanPy: Software for the analysis and visualization of whole-cell current-clamp recordings.

The analysis of action potentials and other membrane voltage fluctuations provides a powerful approach for interrogating the function of excitable cells. However, a major bottleneck in the interpretation of this critical data is the lack of intuitive, agreed-upon software tools for its analysis. Here, we present SanPy, an open-source and freely available software package for the analysis and exploration of whole-cell current-clamp recordings written in Python. SanPy provides a robust computational engine with an application programming interface. Using this, we have developed a cross-platform desktop application with a graphical user interface that does not require programming. SanPy is designed to extract common parameters from action potentials, including threshold time and voltage, peak, half-width, and interval statistics. In addition, several cardiac parameters are measured, including the early diastolic duration and rate. SanPy is built to be fully extensible by providing a plugin architecture for the addition of new file loaders, analysis, and visualizations. A key feature of SanPy is its focus on quality control and data exploration. In the desktop interface, all plots of the data and analysis are linked, allowing simultaneous data visualization from different dimensions with the goal of obtaining ground-truth analysis. We provide documentation for all aspects of SanPy, including several use cases and examples. To test SanPy, we performed analysis on current-clamp recordings from heart and brain cells. Taken together, SanPy is a powerful tool for whole-cell current-clamp analysis and lays the foundation for future extension by the scientific community.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

SanPy: A whole-cell electrophysiology analysis pipeline.

The analysis of action potentials and other membrane voltage fluctuations provide a powerful approach for interrogating the function of excitable cells. Yet, a major bottleneck in the interpretation of this critical data is the lack of intuitive, agreed upon software tools for its analysis. Here, we present SanPy, a Python-based open-source and freely available software pipeline for the analysis and exploration of whole-cell current-clamp recordings. SanPy provides a robust computational engine with an application programming interface. Using this, we have developed a cross-platform graphical user interface that does not require programming. SanPy is designed to extract common parameters from action potentials including threshold time and voltage, peak, half-width, and interval statistics. In addition, several cardiac parameters are measured including the early diastolic duration and rate. SanPy is built to be fully extensible by providing a plugin architecture for the addition of new file loaders, analysis, and visualizations. A key feature of SanPy is its focus on quality control and data exploration. In the desktop interface, all plots of the data and analysis are linked allowing simultaneous data visualization from different dimensions with the goal of obtaining ground truth analysis. We provide documentation for all aspects of SanPy including several use cases and examples. To test SanPy, we have performed analysis on current-clamp recordings from heart and brain cells. Taken together, SanPy is a powerful tool for whole-cell current-clamp analysis and lays the foundation for future extension by the scientific community.

Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

NaV1.1 is essential for proprioceptive signaling and motor behaviors.

The voltage-gated sodium channel (NaV), NaV1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for NaV1.1 in mammalian proprioception. RNAscope analysis and in vitro patch-clamp recordings in genetically identified mouse proprioceptors show ubiquitous channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of NaV1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting that this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings from conditional knockout mice found that loss of NaV1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other NaV isoforms is sufficient to elicit activity in response to transient stimuli, NaV1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, NaV1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest that sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which NaV1.1 function is compromised. Collectively, we present the first evidence that NaV1.1 is essential for mammalian proprioceptive signaling and behaviors.

The mechanisms of cold encoding.

Cold sensation is initiated in the periphery by a specialized population of cold-sensitive neurons, referred to as cold receptors, who transmit decreases in temperature with sub-degree resolution using a diverse assortment of ion channels and receptors. It is largely accepted that normal cold signaling is initiated through activation of transient receptor potential melastatin 8 (TRPM8) expressing neurons. Conversely, the mechanisms underlying cold-induced pain signaling are not as well defined. Interestingly, mounting evidence demonstrates functional interplay between cold signaling and other somatic sensations, such as itch and warmth; thus, cold-sensing pathways also engage in sensory crosstalk and population coding mechanisms. In this review, we will discuss recent advances in our understanding of cold sensation and address major gaps in knowledge that require more investigation.

Mechanisms of ATP release in pain: role of pannexin and connexin channels.

Pain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing.

Tetrodotoxin-Sensitive Sodium Channels Mediate Action Potential Firing and Excitability in Menthol-Sensitive Vglut3-Lineage Sensory Neurons.

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

Merkel Cells Activate Sensory Neural Pathways through Adrenergic Synapses.

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of ß2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT.

Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2.

KEY POINTS: Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability. KARs associate with the auxiliary proteins neuropilin- and tolloid-like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date. M3-S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner. We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity. We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR-specific modulators to alter receptor function. ABSTRACT: Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit- and Neto isoform-specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3-S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3-S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand-binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3-S2 linkers, thereby correlating the action of Neto2 at these structurally discrete sites on receptor subunits. These results demonstrate that the KAR M3-S2 linkers and LBD dimer interface are critical determinants for Neto2 modulation of receptor function and identify these domains as potential sites of action for the targeted development of KAR-specific modulators that alter the function of auxiliary proteins in native receptors.

Coordinated nuclear and synaptic shuttling of afadin promotes spine plasticity and histone modifications.

The ability of a neuron to transduce extracellular signals into long lasting changes in neuronal morphology is central to its normal function. Increasing evidence shows that coordinated regulation of synaptic and nuclear signaling in response to NMDA receptor activation is crucial for long term memory, synaptic tagging, and epigenetic signaling. Although mechanisms have been proposed for synapse-to-nuclear communication, it is unclear how signaling is coordinated at both subcompartments. Here, we show that activation of NMDA receptors induces the bi-directional and concomitant shuttling of the scaffold protein afadin from the cytosol to the nucleus and synapses. Activity-dependent afadin nuclear translocation peaked 2 h post-stimulation, was independent of protein synthesis, and occurred concurrently with dendritic spine remodeling. Moreover, activity-dependent afadin nuclear translocation coincides with phosphorylation of histone H3 at serine 10 (H3S10p), a marker of epigenetic modification. Critically, blocking afadin nuclear accumulation attenuated activity-dependent dendritic spine remodeling and H3 phosphorylation. Collectively, these data support a novel model of neuronal nuclear signaling whereby dual-residency proteins undergo activity-dependent bi-directional shuttling from the cytosol to synapses and the nucleus, coordinately regulating dendritic spine remodeling and histone modifications.

Cyclohexanol analogues are positive modulators of GABA(A) receptor currents and act as general anaesthetics in vivo.

GABA(A) receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanols were investigated on recombinant human γ-aminobutyric acid (GABA(A), α(1)ß(2)γ(2s)) receptors expressed in Xenopus oocytes, and compared to the modulatory effects on GABA currents observed with exposures to the intravenous anaesthetic agent, propofol. Submaximal EC(20) GABA currents were typically enhanced by co-applications of 3-300 µM cyclohexanols. For instance, at 30 µM 2,6-diisopropylcyclohexanol (a novel compound) GABA responses were increased ~3-fold (although similar enhancements were achieved at 3 µM propofol). As regards rank order for modulation by the cyclohexanol analogues at 30 µM, the % enhancements for 2,6-dimethylcyclohexanol~2,6-diethylcyclohexanol~2,6-diisopropylcyclohexanol~2,6-di-sec-butylcyclohexanol ≫2,6-di-tert-butylcyclohexanol~4-tert-butylcyclohexanol>cyclohexanol~cyclopentanol~2-methylcyclohexanol. We further tested the potencies of the cyclohexanol analogues as general anaesthetics using a tadpole in vivo assay. Both 2,6-diisopropylcyclohexanol and 2,6-dimethylcyclohexanol were effective as anaesthetics with EC(50)s of 14.0 µM and 13.1 µM respectively, while other cyclohexanols with bulkier side chains were less potent. In conclusion, our data indicate that cyclohexanols are both positive modulators of GABA(A) receptors currents and anaesthetics. The positioning and size of the alkyl groups at the 2 and 6 positions on the cyclohexanol ring were critical determinants of activity.

Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol.

Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol >> cyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor.