Somatosensation connects animals to their immediate environment, shaping critical behaviors essential for adaptation, learning, and survival. Probing the relationships between somatosensory inputs and behavior in mice presents substantial challenges, primarily due to the practical difficulties of delivering stimuli to the skin during movement. To address this problem, we have developed a system for precise cutaneous stimulation of mice as they walk and run through environments. The system employs real-time body part tracking and targeted optical stimuli, offering precision while preserving the naturalistic context of the behaviors studied, thereby overcoming the traditional trade-offs between precision and animal behavior. We demonstrate the system from nociceptive testing conducted in standard small chambers to behavior in large complex environments, such as mazes. We observed that cutaneous inputs evoke rapid responses, which modify behavior when stimuli are applied during motion. This system provides a means to explore the diverse and integrative nature of somatosensation, from reflexes to decision-making, in naturalistic settings.

Somatosensory stimuli guide and shape behavior, from immediate protective reflexes to longer-term learning and higher-order processes related to pain and touch. However, somatosensory inputs are challenging to control in awake mammals due to the diversity and nature of contact stimuli. Application of cutaneous stimuli is currently limited to relatively imprecise methods as well as subjective behavioral measures. The strategy we present here overcomes these difficulties, achieving 'remote touch' with spatiotemporally precise and dynamic optogenetic stimulation by projecting light to a small defined area of skin. We mapped behavioral responses in freely behaving mice with specific nociceptor and low-threshold mechanoreceptor inputs. In nociceptors, sparse recruitment of single-action potentials shapes rapid protective pain-related behaviors, including coordinated head orientation and body repositioning that depend on the initial body pose. In contrast, activation of low-threshold mechanoreceptors elicited slow-onset behaviors and more subtle whole-body behaviors. The strategy can be used to define specific behavioral repertoires, examine the timing and nature of reflexes, and dissect sensory, motor, cognitive, and motivational processes guiding behavior.

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

Activation of nociceptor sensory neurons by noxious stimuli both triggers pain and increases capillary permeability and blood flow to produce neurogenic inflammation(1,2), but whether nociceptors also interact with the immune system remains poorly understood. Here we report a neurotechnology for selective epineural optogenetic neuromodulation of nociceptors and demonstrate that nociceptor activation drives both protective pain behavior and inflammation. The wireless optoelectronic system consists of sub-millimeter-scale light-emitting diodes embedded in a soft, circumneural sciatic nerve implant, powered and driven by a miniaturized head-mounted control unit. Photostimulation of axons in freely moving mice that express channelrhodopsin only in nociceptors resulted in behaviors characteristic of pain, reflecting orthodromic input to the spinal cord. It also led to immune reactions in the skin in the absence of inflammation and potentiation of established inflammation, a consequence of the antidromic activation of nociceptor peripheral terminals. These results reveal a link between nociceptors and immune cells, which might have implications for the treatment of inflammation. An optogenetic study shows that activation of pain-triggering sensory neurons induces an immune response.

Objective. Nerves in the peripheral nervous system (PNS) contain axons with specific motor, somatosensory and autonomic functions. Optogenetics offers an efficient approach to selectively activate axons within the nerve. However, the heterogeneous nature of nerves and their tortuous route through the body create a challenging environment to reliably implant a light delivery interface. Approach. Here, we propose an optical peripheral nerve interface-an optocuff-, so that optogenetic modulation of peripheral nerves become possible in freely behaving mice. Main results. Using this optocuff, we demonstrate orderly recruitment of motor units with epineural optical stimulation of genetically targeted sciatic nerve axons, both in anaesthetized and in awake, freely behaving animals. Behavioural experiments and histology show the optocuff does not damage the nerve thus is suitable for long-term experiments. Significance. These results suggest that the soft optocuff might be a straightforward and efficient tool to support more extensive study of the PNS using optogenetics.

Potentially harmful stimuli are detected at the skin by nociceptor sensory neurons that drive rapid protective withdrawal reflexes and pain. We set out to define, at a millisecond timescale, the relationship between the activity of these sensory neurons and the resultant behavioral output. Brief optogenetic activation of cutaneous nociceptors was found to activate only a single action potential in each fiber. This minimal input was used to determine high-speed behavioral responses in freely behaving mice. The localized stimulus generated widespread dynamic repositioning and alerting sub-second behaviors whose nature and timing depended on the context of the animal and its position, activity, and alertness. Our findings show that the primary response to injurious stimuli is not limited, fixed, or localized, but is dynamic, and that it involves recruitment and gating of multiple circuits distributed throughout the central nervous system at a sub-second timescale to effectively both alert to the presence of danger and minimize risk of harm.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Background: The ATP-gated channel (P2X2) receptor ectodomain is formed by β-strands from three subunits. Results: ATP-induced channel opening was prevented by disulfide locking between pairs of substituted cysteines in different subunits. Conclusion: The turret region, the lower body wall, and the outer ends of transmembrane domains move significantly in channel opening. Significance: Large ectodomain movements couple ATP binding to channel opening in P2X receptors. The ectodomain of the P2X receptor is formed mainly from two- or three-stranded β-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the β-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.

Optogenetic methods to control nociceptors open up a wealth of potential applications in pain research and therapy.

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2′(3′)-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.

P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.

The selectivity of ion channels is generally considered to be fixed. The ATP-gated P2X7 receptor is unusual in that its activation leads to a progressive increase in the permeation of large cations during several seconds (e.g. YO-PRO-1). However, controversy remains whether these large molecules directly permeate the P2X7 receptor pore or use some coupled transport mechanism. In HEK293 cells expressing the rP2X7 receptor, simultaneous measurement of ionic current and YO-PRO-1 influx showed they both increased concomitantly with the application of ATP. At positive potentials YO-PRO-1 influx was much reduced, implying that movement of this large cation (1.8 x 0.8 x 0.7 nm) is influenced by the membrane electric field in a manner similar to smaller cations. Increasing the positivity in the pore of the P2X7 receptor significantly enhanced chloride permeability (D352N and T348K; PCl/PNa increased 10-fold compared to WT). The selective permeability of large molecules was then measured using two ions of similar size and structure, bearing either a positive or a negative net charge: both the cation (ethidium: 1.2 x 1.0 x 0.5 nm) and the anion (FITC: 1.3 x 1.1 x 0.7 nm) were simultaneously detected by intracellular fluorescence. Introduction of a positive charge in the pore (T348K) or removal of a negative charge (D352N) increased the influx of the anion FITC, and decreased the influx of the cation ethidium. These effects parallel the findings of chloride permeability, demonstrating that by increasing the positivity in the pore of the P2X7 receptor the permeation pathway is more energetically favourable for anions, whether they are small or large. This implies that large molecules directly permeate the P2X7 receptor pore and any model of channel opening should accommodate a pore diameter >1 nm.

P2X receptors are trimeric ion channels that open in response to extracellular ATP. The publication of the first X-ray structure of a closed P2X receptor has confirmed many of the functional experiments carried out since the mid-1990s and provides a point of reference for the molecular dissection of P2X receptor function. These co-ordinates shed light on the ATP binding sites housed in the extracellular domain, the contacts between subunits, the pathways for ion access, and the cation-selective pore that spans the cell membrane. They also provide a template for structure-based design of much needed pharmacological agents to act on a receptor superfamily whose physiological roles extend from synaptic transmission to inflammation and control of programmed cell death. Interpretation of functional data in the context of this closed P2X receptor structure reveals structural rearrangements in the transmembrane pore and extracellular domain. Here the current understanding of the molecular structure of P2X receptors is reviewed. This review will be of relevance to those interested in the mechanisms that underlie the molecular operation of P2X receptors, and other ion channels gated by diffusible ligands (pentameric Cys-loop receptors, tetrameric glutamate receptors), as well as trimeric ion channels that are distantly related to P2X receptors (acid-sensing ion channels and the epithelial sodium channel). WIREs Membr Transp Signal 2012, 1:56–69. doi: 10.1002/wmts.24 For further resources related to this article, please visit the WIREs website .

BACKGROUND AND PURPOSE The human P2X7 (hP2X7) receptor exhibits striking pharmacological differences from its rodent counterparts, particularly in terms of its antagonist profile. Here, we characterized the functional and pharmacological properties of the rhesus macaque monkey P2X7 (rmP2X7) receptor in comparison with the hP2X7 receptor.

P2X receptors are ATP-gated membrane ion channels with multifarious roles, including afferent sensation, autocrine feedback loops, and inflammation. Their molecular operation has been less well elucidated compared with other ligand-gated channels (nicotinic acetylcholine receptors, ionotropic glutamate receptors). This will change with the recent publication of the crystal structure of a closed P2X receptor. Here we re-interpret results from 15 years of experiments using site-directed mutagenesis with a model based on the new structure. Previous predictions of receptor stoichiometry, the extracellular ATP binding site, inter-subunit contacts, and many details of the permeation pathway fall into place in three dimensions. We can therefore quickly understand how the channel operates at the molecular level. This is important not only for ion- channel aficionados, but also those engaged in developing effective antagonists at P2X receptors for potential therapeutic use.

P2X receptor channels, which have three α-helical transmembrane domains, are found throughout the nervous system. Using site-directed mutagenesis, the authors find that each of the three subunits contributes equally to the open channel permeation pathway and that the closed-to-open transition involves a symmetrical separation of the three channel subunits. In the closed structure of the P2X cation channel, three α-helical transmembrane domains cross the membrane obliquely. In rat P2X2 receptors, these intersect at Thr339. Replacing Thr339 by lysine in one, two or three subunits progressively increased chloride permeability and reduced unitary conductance. This implies that the closed-open transition involves a symmetrical separation of the three subunits and that Thr339 from each subunit contributes symmetrically to the open channel permeation pathway.

The aim of this work is to characterise the functional properties of human and rat Na(V)1.8 channels and to investigate the action of anti-nociceptive agents. Na(V)1.8 alpha-subunits were expressed in mammalian sensory neuron-derived ND7/23 cells, and sodium currents were recorded using whole-cell patch clamp. The current-voltage curves for activation were similar for human and rat Na(V)1.8 channels. However, for inactivation, human Na(V)1.8 showed more hyperpolarised voltage-dependence than for the rat channel, faster development of inactivation, slower recovery from the fast component of inactivation, and faster recovery from the slow component. Thus, this would imply that the human channel is more inactivated at normal resting potentials. Compounds 227c89, A-803467, V102862, ralfinamide and tetracaine all showed greater affinity for the inactivated state than for the resting state. Compounds A-803467 and V102862 were the most potent, and A-803467 showed greater inactivated state affinity for human than for rat channels. Surprisingly, during recovery from inactivation, an increase in current was observed for V102862 and A-803467, probably due to disinhibition of resting block. Rather than the use-dependent inhibition normally seen with inactivated state blockers, for A-803467 this disinhibition led to an increase in current during repetitive stimulation, while V102862 showed less inhibition than otherwise expected at lower frequencies. Thus the data supports the suggestion that, while both V102862 and A-803467 are potent inhibitors of Na(V)1.8, the compound V102862, rather than A-803467, may be useful as an analgesic where physiological firing frequencies are higher.