Primary Somatosensory Neurons Research Articles

Optogenetic Activation of Peripheral Somatosensory Neurons in Transgenic Mice as a Neuropathic Pain Model for Assessing the Therapeutic Efficacy of Analgesics.

Optogenetics is a novel biotechnology widely used to precisely manipulate a specific peripheral sensory neuron or neural circuit. However, the use of optogenetics to assess the therapeutic efficacy of analgesics is elusive. In this study, we generated a transgenic mouse stain in which all primary somatosensory neurons can be optogenetically activated to mimic neuronal hyperactivation in the neuropathic pain state for the assessment of analgesic effects of drugs. A transgenic mouse was generated using the advillin-Cre line mated with the Ai32 strain, in which channelrhodopsin-2 fused to enhanced yellow fluorescence protein (ChR2-EYFP) was conditionally expressed in all types of primary somatosensory neurons (advillincre/ChR2+/+). Immunofluorescence and transdermal photostimulation on the hindpaws were used to verify the transgenic mice. Optical stimulation to evoke pain-like paw withdrawal latency was used to assess the analgesic effects of a series of drugs. Injury- and pain-related molecular biomarkers were investigated with immunohistofluorescence. We found that the expression of ChR2-EYFP was observed in many primary afferents of paw skin and sciatic nerves and in primary sensory neurons and laminae I and II of the spinal dorsal horns in advillincre/ChR2+/+ mice. Transdermal blue light stimulation of the transgenic mouse hindpaw evoked nocifensive paw withdrawal behavior. Treatment with gabapentin, some channel blockers, and local anesthetics, but not opioids or COX-1/2 inhibitors, prolonged the paw withdrawal latency in the transgenic mice. The analgesic effect of gabapentin was also verified by the decreased expression of injury- and pain-related molecular biomarkers. These optogenetic mice provide a promising model for assessing the therapeutic efficacy of analgesics in neuropathic pain.

Targeting TRPV1+ primary somatosensory neurons for modulating pain and itch sensations

Targeting TRPV1+ primary somatosensory neurons for modulating pain and itch sensations

Thalamocortical circuits drive remifentanil-induced postoperative hyperalgesia

Remifentanil-induced hyperalgesia (RIH) is a severe but common postoperative clinical problem with elusive underlying neural mechanisms. Here, we discovered that glutamatergic neurons in the thalamic ventral posterolateral nucleus (VPLGlu) exhibited significantly elevated burst firing accompanied by upregulation of Cav3.1 T-type calcium channel expression and function in RIH model mice. In addition, we identified a glutamatergic neuronal thalamocortical circuit in the VPL projecting to hindlimb primary somatosensory cortex glutamatergic neurons (S1HLGlu) that mediated RIH. In vivo calcium imaging and multi-tetrode recordings revealed heightened S1HLGlu neuronal activity during RIH. Moreover, preoperative suppression of Cav3.1-dependent burst firing in VPLGlu neurons or chemogenetic inhibition of VPLGlu neuronal terminals in the S1HL abolished the increased S1HLGlu neuronal excitability while alleviating RIH. Our findings suggest that remifentanil induces postoperative hyperalgesia by upregulating T-type calcium channel-dependent burst firing in VPLGlu neurons to activate S1HLGlu neurons, thus revealing an ion channel-mediated neural circuit basis for RIH that can guide analgesic development.

Behaviorally relevant decision coding in primary somatosensory cortex neurons.

Primary sensory cortex is thought to process incoming sensory information, while decision variables important for driving behavior are assumed to arise downstream in the processing hierarchy. We used population two-photon calcium imaging and targeted two-photon optogenetic stimulation of neurons in layer 2/3 of mouse primary somatosensory cortex (S1) during a texture discrimination task to test for the presence of decision signals and probe their behavioral relevance. Small but distinct populations of neurons carried information about the stimulus irrespective of the behavioral outcome (‘stimulus neurons’), or about the choice irrespective of the presented stimulus (‘decision neurons’). Decision neurons show categorical coding that develops during learning, and lack a conclusive decision signal in miss trials. All-optical photostimulation of decision neurons improves behavioral performance, establishing a causal role in driving behavior. The fact that stimulus and decision neurons are intermingled challenges the idea of S1 as a purely sensory area, and causal perturbation suggests a direct involvement of S1 decision neurons in the decision-making process.

Oh, What a Tangled Web Epilepsy Genes May Weave.

Aberrant Neuronal Connectivity in the Cortex Drives Generation of Seizures in Rat Absence Epilepsy Studer F, Jarre G, Pouyatos B, et al. Brain 2022;145(6):1978-1991. doi:10.1093/brain/awab438.Absence epilepsy belongs to genetic epilepsies and is characterized by recurrent generalized seizures that are concomitant with alterations of consciousness and associated with cognitive comorbidities. Little is known about the mechanisms leading to occurrence of epileptic seizures (i.e., epileptogenesis) and, in particular, it remains an open question whether neuronal hypersynchronization, a key feature in seizure initiation, could result from aberrant structural connectivity within neuronal networks endowing them with epileptic properties. In the present study, we addressed this question using a genetic model of absence epilepsy in the rat where seizures initiate in the whisker primary somatosensory cortex. We hypothesized that alterations in structural connectivity of neuronal networks within wS1 contribute to pathological neuronal synchronization responsible for seizures. First, we used rabies virus-mediated retrograde synaptic tracing and evidenced that cortical neurons located in both upper- and deep-layers of whisker primary somatosensory cortex displayed aberrant and significantly increased connectivity the genetic model of absence epilepsy, as highlighted by a higher number of presynaptic partners. Next, we showed at the functional level that disrupting these aberrant whisker primary somatosensory cortex neuronal networks with synchrotron X-ray-mediated cortical microtransections drastically decreased both whisker primary somatosensory cortex neuron synchronization and seizure power, as revealed by in vivo local field potential recordings with multichannel probes. Taken together, our data provide for the first time strong evidence that increased structural connectivity patterns of cortical neurons represent critical pathological substrates for increased neuronal synchronization and generation of absence seizures.

Illusory tactile movement crosses arms and legs and is coded in external space

Humans often misjudge where on the body a touch occurred. Theoretical accounts have ascribed such misperceptions to local interactions in peripheral and primary somatosensory neurons, positing that spatial-perceptual mechanisms adhere to limb boundaries and skin layout. Yet, perception often reflects integration of sensory signals with prior experience. On their trajectories, objects often touch multiple limbs; therefore, body–environment interactions should manifest in perceptual mechanisms that reflect external space.Here, we demonstrate that humans perceived the cutaneous rabbit illusion – the percept of multiple identical stimuli as hopping across the skin – along the Euclidian trajectory between stimuli on two body parts and regularly mislocalized stimuli from one limb to the other. A Bayesian model based on Euclidian, as opposed to anatomical, distance faithfully reproduced key aspects of participants' localization behavior.Our results suggest that prior experience of touch in space critically shapes tactile spatial perception and illusions beyond anatomical organization.

Trehalose Reduces Nerve Injury Induced Nociception in Mice but Negatively Affects Alertness

Trehalose, a sugar from fungi, mimics starvation due to a block of glucose transport and induces Transcription Factor EB- mediated autophagy, likely supported by the upregulation of progranulin. The pro-autophagy effects help to remove pathological proteins and thereby prevent neurodegenerative diseases such as Alzheimer’s disease. Enhancing autophagy also contributes to the resolution of neuropathic pain in mice. Therefore, we here assessed the effects of continuous trehalose administration via drinking water using the mouse Spared Nerve Injury model of neuropathic pain. Trehalose had no effect on drinking, feeding, voluntary wheel running, motor coordination, locomotion, and open field, elevated plus maze, and Barnes Maze behavior, showing that it was well tolerated. However, trehalose reduced nerve injury-evoked nociceptive mechanical and thermal hypersensitivity as compared to vehicle. Trehalose had no effect on calcium currents in primary somatosensory neurons, pointing to central mechanisms of the antinociceptive effects. In IntelliCages, trehalose-treated mice showed reduced activity, in particular, a low frequency of nosepokes, which was associated with a reduced proportion of correct trials and flat learning curves in place preference learning tasks. Mice failed to switch corner preferences and stuck to spontaneously preferred corners. The behavior in IntelliCages is suggestive of sedative effects as a “side effect” of a continuous protracted trehalose treatment, leading to impairment of learning flexibility. Hence, trehalose diet supplements might reduce chronic pain but likely at the expense of alertness.

Distinct thalamocortical circuits underlie allodynia induced by tissue injury and by depression-like states.

In humans, tissue injury and depression can both cause pain hypersensitivity, but whether this involves distinct circuits remains unknown. Here, we identify two discrete glutamatergic neuronal circuits in male mice: a projection from the posterior thalamic nucleus (POGlu) to primary somatosensory cortex glutamatergic neurons (S1Glu) mediates allodynia from tissue injury, whereas a pathway from the parafascicular thalamic nucleus (PFGlu) to anterior cingulate cortex GABA-containing neurons to glutamatergic neurons (ACCGABA→Glu) mediates allodynia associated with a depression-like state. In vivo calcium imaging and multi-tetrode electrophysiological recordings reveal that POGlu and PFGlu populations undergo different adaptations in the two conditions. Artificial manipulation of each circuit affects allodynia resulting from either tissue injury or depression-like states, but not both. Our study demonstrates that the distinct thalamocortical circuits POGlu→S1Glu and PFGlu→ACCGABA→Glu subserve allodynia associated with tissue injury and depression-like states, respectively, thus providing insights into the circuit basis of pathological pain resulting from different etiologies.

Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics.

Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with non-neuronal cell types in sensory ganglia. We describe a protocol to isolate nuclei from mouse DRGs using iodixanol density gradient centrifugation, which enriches for neuronal nuclei while still sampling non-neuronal cells such as satellite glia and Schwann cells. This protocol is compatible with a range of downstream applications such as single-nucleus transcriptional and epigenomic assays.

Pricking into Autonomic Reflex Pathways by Electrical Acupuncture

Using electrical acupuncture, Liu etal. show how electrical stimulation of primary somatosensory neurons at different body regions can tap into discreet autonomic circuits and, depending on the parameters, initiate either a pro- or anti-inflammatory response.

Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury

Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.

TRESK and TREK-2 two-pore-domain potassium channel subunits form functional heterodimers in primary somatosensory neurons

Two-pore-domain potassium channels (K2P) are the major determinants of the background potassium conductance. They play a crucial role in setting the resting membrane potential and regulating cellular excitability. These channels form homodimers; however, a few examples of heterodimerization have also been reported. The K2P channel subunits TRESK and TREK-2 provide the predominant background potassium current in the primary sensory neurons of the dorsal root and trigeminal ganglia. A recent study has shown that a TRESK mutation causes migraine because it leads to the formation of a dominant negative truncated TRESK fragment. Surprisingly, this fragment can also interact with TREK-2. In this study, we determined the biophysical and pharmacological properties of the TRESK/TREK-2 heterodimer using a covalently linked TRESK/TREK-2 construct to ensure the assembly of the different subunits. The tandem channel has an intermediate single-channel conductance compared with the TRESK and TREK-2 homodimers. Similar conductance values were recorded when TRESK and TREK-2 were coexpressed, demonstrating that the two subunits can spontaneously form functional heterodimers. The TRESK component confers calcineurin-dependent regulation to the heterodimer and gives rise to a pharmacological profile similar to the TRESK homodimer, whereas the presence of the TREK-2 subunit renders the channel sensitive to the selective TREK-2 activator T2A3. In trigeminal primary sensory neurons, we detected single-channel activity with biophysical and pharmacological properties similar to the TRESK/TREK-2 tandem, indicating that WT TRESK and TREK-2 subunits coassemble to form functional heterodimeric channels also in native cells.

The emergence of transcriptional identity in somatosensory neurons

More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments1-4. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific geneexpression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.

Effects of ketamine on voltage-gated sodium channels in the barrel cortex and the ventral posteromedial nucleus slices of rats.

Ketamine is commonly used as a dissociative anesthetic with unique actions in the central nervous system. Previous studies have found that the thalamocortical systems play an important role in general anesthetics induced unconsciousness. Whether the voltage-gated sodium channels in the thalamocortical systems are the target of ketamine remain unclear. The present study used a whole-cell patch-clamp technique to observe the effects of ketamine on voltage-gated Na channels in thalamocortical pyramidal neurons. We found that IC50 of ketamine on Na currents in the primary somatosensory barrel cortex pyramidal neurons and the thalamus ventral posteromedial nucleus pyramidal neurons was 686.72 ± 39.92 and 842.65 ± 87.28 μM, respectively. Ketamine accelerated the Na channels inactivation and slowed inactivation of Na channels after recovery but did not affect the activation. We demonstrated the detailed suppression process of neural voltage-gated Na channels by ketamine on thalamocortical slice. This may provide a new insight into the mechanical explanation for the ketamine anesthesia.

(345) The Neuropeptide TLQP-62 Induces Calcium Transients in Primary Afferent Neurons

The neurosecretory protein VGF (non-acronymic) is rapidly and robustly upregulated by primary somatosensory afferent neurons following peripheral nerve damage. VGF is proteolytically processed to yield a number of bioactive peptides, including the C-terminal peptide TLQP-62. Our work demonstrates that spinal TLQP-62 increases after nerve injury and that immunoneutralization of C-terminal VGF peptides attenuates the development of mechanical allodynia. This peptide has been shown to induce BDNF-dependent neuronal plasticity in the hippocampus. We have shown that TLQP-62 induces potentiation of glutamatergic responses in a subset of dorsal horn neurons and exposure of spinal cord slices to TLQP-62 increases TrkB phosphorylation, suggesting that TLQP-62 may facilitate BDNF release. Since primary afferent neurons are a potential source of spinal BDNF, we examined the effects of TLQP-62 on these neurons. Using mice that express a genetically encoded calcium indicator restricted to primary afferent neurons (Pirt-GCamp3 mice), we observed that TLQP-62 induced calcium transients in a subset of primary afferent fibers in the dorsal horn of spinal cord slices. The responses occurred immediately after TLQP-62 exposure and were sustained during prolonged exposure. TLQP-62 also induced calcium transients in DRG cultures, both in capsaicin-responsive neurites, as well as in neurites that did not respond to capsaicin. Ongoing work is investigating the functional consequences of these TLQP-62 effects and their relationship to BDNF via patch-clamp and BDNF-pHluorin imaging, respectively. Our observations of TLQP-62-induced calcium responses in the processes of primary afferent neurons are consistent with the idea that the peptide may modulate neurotransmitter release.

Critical role of the pore domain in the cold response of TRPM8 channels identified by ortholog functional comparison

In mammals, the main molecular entity involved in innocuous cold transduction is TRPM8. This polymodal ion channel is activated by cold, cooling compounds such as menthol and voltage. Despite its relevance, the molecular determinants involved in its activation by cold remain elusive. In this study we explored the use of TRPM8 orthologs with different cold responses as a strategy to identify new molecular determinants related with their thermosensitivity. We focused on mouse TRPM8 (mTRPM8) and chicken TRPM8 (cTRPM8), which present complementary thermosensitive and chemosensitive phenotypes. Although mTRPM8 displays larger responses to cold than cTRPM8 does, the avian ortholog shows a higher sensitivity to menthol compared with the mouse channel, in both HEK293 cells and primary somatosensory neurons. We took advantage of these differences to build multiple functional chimeras between these orthologs, to identify the regions that account for these discrepancies. Using a combination of calcium imaging and patch clamping, we identified a region encompassing positions 526-556 in the N terminus, whose replacement by the cTRPM8 homolog sequence potentiated its response to agonists. More importantly, we found that the characteristic cold response of these orthologs is due to nonconserved residues located within the pore loop, suggesting that TRPM8 has evolved by increasing the magnitude of its cold response through changes in this region. Our results reveal that these structural domains are critically involved in cold sensitivity and functional modulation of TRPM8, and support the idea that the pore domain is a key molecular determinant in temperature responses of this thermo-transient receptor potential (TRP) channel.

Using dynamic clamp to quantify pathological changes in the excitability of primary somatosensory neurons.

Key points Primary somatosensory neurons normally respond to somatic depolarization with transient spiking but can switch to repetitive spiking under pathological conditions. This switch in spiking pattern reflects a qualitative change in spike initiation dynamics and contributes to the hyperexcitability associated with chronic pain.Neurons can be converted to repetitive spiking by adding a virtual conductance using dynamic clamp. By titrating the conductance to determine how much must be added to cause repetitive spiking, we found that small cells are more susceptible to switching (i.e. required less added conductance) than medium–large cells.By measuring how much less conductance is required to cause repetitive spiking when dynamic clamp was combined with other pathomimetic manipulations (e.g. application of inflammatory mediators), we measured how much each manipulation facilitated repetitive spiking.Our results suggest that many pathological factors facilitate repetitive spiking but that the switch to repetitive spiking requires the cumulative effect of many co‐occurring factors. Primary somatosensory neurons become hyperexcitable in many chronic pain conditions. Hyperexcitability can include a switch from transient to repetitive spiking during sustained somatic depolarization. This switch results from diverse pathological processes that impact ion channel expression or function. Because multiple pathological processes co‐occur, isolating how much each contributes to switching the spiking pattern is difficult. Our approach to this challenge involves adding a virtual sodium conductance via dynamic clamp. The magnitude of that conductance was titrated to determine the minimum required to enable rheobasic stimulation to evoke repetitive spiking. The minimum required conductance, termed g¯ Na ∗, was re‐measured before and during manipulations designed to model various pathological processes in vitro. The reduction in g¯ Na ∗ caused by each pathomimetic manipulation reflects how much the modelled process contributes to switching the spiking pattern. We found that elevating extracellular potassium or applying inflammatory mediators reduced g¯ Na ∗ whereas direct hyperpolarization had no effect. Inflammatory mediators reduced g¯ Na ∗ more in medium–large (>30 μm diameter) neurons than in small (⩽30 μm diameter) neurons, but had equivalent effects in cutaneous and muscle afferents. The repetitive spiking induced by dynamic clamp was also found to differ between small and medium–large neurons, thus revealing latent differences in adaptation. Our study demonstrates a novel way to determine to what extent individual pathological factors facilitate repetitive spiking. Our results suggest that most factors facilitate but do not cause repetitive spiking on their own, and, therefore, that a switch to repetitive spiking results from the cumulative effect of many co‐occurring factors.

The Glutamatergic System in Primary Somatosensory Neurons and Its Involvement in Sensory Input-Dependent Plasticity.

Glutamate is the most common neurotransmitter in both the central and the peripheral nervous system. Glutamate is present in all types of neurons in sensory ganglia, and is released not only from their peripheral and central axon terminals but also from their cell bodies. Consistently, these neurons express ionotropic and metabotropic receptors, as well as other molecules involved in the synthesis, transport and release of the neurotransmitter. Primary sensory neurons are the first neurons in the sensory channels, which receive information from the periphery, and are thus key players in the sensory transduction and in the transmission of this information to higher centers in the pathway. These neurons are tightly enclosed by satellite glial cells, which also express several ionotropic and metabotropic glutamate receptors, and display increases in intracellular calcium accompanying the release of glutamate. One of the main interests in our group has been the study of the implication of the peripheral nervous system in sensory-dependent plasticity. Recently, we have provided novel evidence in favor of morphological changes in first- and second-order neurons of the trigeminal system after sustained alterations of the sensory input. Moreover, these anatomical changes are paralleled by several molecular changes, among which those related to glutamatergic neurotransmission are particularly relevant. In this review, we will describe the state of the art of the glutamatergic system in sensory ganglia and its involvement in input-dependent plasticity, a fundamental ground for advancing our knowledge of the neural mechanisms of learning and adaptation, reaction to injury, and chronic pain.

Pharmacology of the Nav1.1 domain IV voltage sensor reveals coupling between inactivation gating processes

The Nav1.1 voltage-gated sodium channel is a critical contributor to excitability in the brain, where pathological loss of function leads to such disorders as epilepsy, Alzheimer's disease, and autism. This voltage-gated sodium (Nav) channel subtype also plays an important role in mechanical pain signaling by primary afferent somatosensory neurons. Therefore, pharmacologic modulation of Nav1.1 represents a potential strategy for treating excitability disorders of the brain and periphery. Inactivation is a complex aspect of Nav channel gating and consists of fast and slow components, each of which may involve a contribution from one or more voltage-sensing domains. Here, we exploit the Hm1a spider toxin, a Nav1.1-selective modulator, to better understand the relationship between these temporally distinct modes of inactivation and ask whether they can be distinguished pharmacologically. We show that Hm1a inhibits the gating movement of the domain IV voltage sensor (VSDIV), hindering both fast and slow inactivation and leading to an increase in Nav1.1 availability during high-frequency stimulation. In contrast, ICA-121431, a small-molecule Nav1.1 inhibitor, accelerates a subsequent VSDIV gating transition to accelerate entry into the slow inactivated state, resulting in use-dependent block. Further evidence for functional coupling between fast and slow inactivation is provided by a Nav1.1 mutant in which fast inactivation removal has complex effects on slow inactivation. Taken together, our data substantiate the key role of VSDIV in Nav channel fast and slow inactivation and demonstrate that these gating processes are sequential and coupled through VSDIV. These findings provide insight into a pharmacophore on VSDIV through which modulation of inactivation gating can inhibit or facilitate Nav1.1 function.

(206) Modulation of multiple modalities of somatosensory information by peripheral kappa opioid receptors

Peripherally-restricted kappa opioid receptor (KOR) agonists are emerging as a novel treatment for pain and itch conditions and have shown efficacy in several recent clinical trials. One primary site of action is thought to be at KORs on primary afferent nerves that innervate the body. Yet, the subtypes of primary afferent somatosensory neurons that express the kappa opioid receptor remain undefined. Using a newly developed KOR-cre knockin allele, viral tracing, and single-cell RT-PCR we discovered that that KOR is expressed in a specific subset of peptidergic afferents. This subset targeted multiple tissue types and expressed higher levels of transcripts known to be involved in neurogenic inflammation. Consistent with this, peripherally-restricted KOR agonists inhibited behavioral responses to chemical pain and itch stimuli, but not acute heat stimuli, and also decreased mechanical hypersensitivity following an incision injury. Unexpectedly, we also found that KOR is expressed in subsets of primary afferents that form lanceolate or circumferential endings around hair follicles, suggesting an unappreciated role for KOR signaling in the modulation of low-threshold mechanosensation. At a functional level, genetically-labeled afferents showed inhibition of voltage-gated calcium current in response to kappa agonists, and optogenetic experiments revealed that dynorphin inhibited evoked-EPSCs from the central terminals of KOR-expressing afferents. These experiments provide key insight for the rationale use of peripherally-restricted KOR agonists for the modulation of inflammatory pain, itch, and potentially mechanical allodynia.