A spinal neural circuitry for converting touch to itch sensation

Dorsal horn gastrin-releasing peptide receptor (GRPR) neurons have a central role in itch transmission. Itch signaling has been suggested to be controlled by an inhibitory network in the spinal dorsal horn, as increased scratching behavior can be induced by pharmacological disinhibition or ablation of inhibitory interneurons, but the direct influence of the inhibitory tone on the GRPR neurons in the itch pathway have not been explored. Here we have investigated spinal GRPR neurons through in vitro and bioinformatical analysis. Electrophysiological recordings revealed that GRPR neurons receive local spontaneous excitatory inputs transmitted by glutamate and inhibitory inputs by glycine and GABA, which were transmitted either by separate glycinergic and GABAergic synapses or by glycine and GABA co-releasing synapses. Additionally, all GRPR neurons received both glycine- and GABA-induced tonic currents. The findings show a complex inhibitory network, composed of synaptic and tonic currents that gates the excitability of GRPR neurons, which provides direct evidence for the existence of an inhibitory tone controlling spontaneous discharge in an itch-related neuronal network in the spinal cord. Finally, calcium imaging revealed increased levels of neuronal activity in Grpr-Cre neurons upon application of somatostatin, which provides direct in vitro evidence for disinhibition of these dorsal horn interneurons.

Socially contagious itch is ubiquitous in human society, but whether it exists in rodents is unclear. Using a behavioral paradigm that does not entail prior training or reward, we found that mice scratched after observing a conspecific scratching. Molecular mapping showed increased neuronal activity in the suprachiasmatic nucleus (SCN) of the hypothalamus of mice that displayed contagious scratching. Ablation of gastrin-releasing peptide receptor (GRPR) or GRPR neurons in the SCN abolished contagious scratching behavior, which was recapitulated by chemogenetic inhibition of SCN GRP neurons. Activation of SCN GRP/GRPR neurons evoked scratching behavior. These data demonstrate that GRP-GRPR signaling is necessary and sufficient for transmitting contagious itch information in the SCN. The findings may have implications for our understanding of neural circuits that control socially contagious behaviors.

SummarySpinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itch-like behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output.

Mechanistic insights into spinal neurones involved in neuraxial opioid-induced pruritus

It has been known that algogens and cooling could inhibit itch sensation; however, the underlying molecular and neural mechanisms remain poorly understood. Here, we show that the spinal neurons expressing gastrin releasing peptide receptor (GRPR) primarily comprise excitatory interneurons that receive direct and indirect inputs from C and Aδ fibers and form contacts with projection neurons expressing the neurokinin 1 receptor (NK1R). Importantly, we show that noxious or cooling agents inhibit the activity of GRPR neurons via GABAergic signaling. By contrast, capsaicin, which evokes a mix of itch and pain sensations, enhances both excitatory and inhibitory spontaneous synaptic transmission onto GRPR neurons. These data strengthen the role of GRPR neurons as a key circuit for itch transmission and illustrate a spinal mechanism whereby pain inhibits itch by suppressing the function of GRPR neurons.

Itch triggers scratching, a behavioural defence mechanism that aids in the removal of harmful irritants and parasites1. Chemical itch is triggered by many endogenous and exogenous cues, such as pro-inflammatory histamine, which is released during an allergic reaction1. Mechanical itch can be triggered by light sensations such as wool fibres or a crawling insect2. In contrast to chemical itch pathways, which have been extensively studied, the mechanisms that underlie the transduction of mechanical itch are largely unknown. Here we show that the mechanically activated ion channel PIEZO1 (ref. 3) is selectively expressed by itch-specific sensory neurons and is required for their mechanically activated currents. Loss of PIEZO1 function in peripheral neurons greatly reduces mechanically evoked scratching behaviours and both acute and chronic itch-evoked sensitization. Finally, mice expressing a gain-of-function Piezo1 allele4 exhibit enhanced mechanical itch behaviours. Our studies reveal the polymodal nature of itch sensory neurons and identify a role for PIEZO1 in the sensation of itch.

A subset of peripheral sensory neurons expressing specific Mas-related G-protein-coupled receptors and transient receptor potential channels mediate pruritogen-induced chemical itch. However, the molecular mechanisms that regulate the excitability of these cells, and consequently itch sensation, are poorly understood. TWIK-related spinal cord K + channel (TRESK) is a background K + channel that modulates the resting membrane potential, action potential firing, and neuronal excitability, and it has been involved in somatosensation and pain transduction. Here, we demonstrate that this channel contributes to pruritic transduction and it is a potential target for treating chronic itch pathologies. TRESK channel coexpress with Mas-related G-protein-coupled receptor A3, MrgprC11 and MrgprD in mouse sensory neurons, and with MrgprX1 in human ones. Genetic ablation of TRESK enhances firing of MrgprA3-expressing pruriceptors and acute itch in response to intradermal injection of chloroquine, while the response to histamine, BAM8-22, or leukotriene C4 remains unaffected. TRESK deletion also exacerbates chronic itch in mouse models of allergic contact dermatitis, dry skin, and imiquimod-induced psoriasiform dermatitis, resulting in a significantly increased scratching behavior that develops earlier and is more robust. Moreover, pharmacologically enhancing TRESK function diminishes both acute and chronic itch in wild-type mice but not in TRESK knockout (KO) animals. In summary, our data indicate that TRESK plays a role in regulating the excitability of a subset of sensory neurons that mediate histaminergic-independent itch. Enhancing the channel function with specific activators represents a promising antipruritic therapeutic approach that can be combined with other compounds for the treatment of nonhistaminergic itch, which currently lack adequate treatment options.

Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75–80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.

We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling.

Molecular taxonomy of nociceptors and pruriceptors.

Alloknesis refers to itch caused by normally non itch-inducing stimuli, particularly light mechanical stimuli, such as contacts with clothes or other human bodies. This symptom occurs in patients suffering from chronic itch. While it has been mainly described in patients with atopic dermatitis, it is probably present in numerous other conditions and it could induce a severe burden. Until now, it is mainly diagnosed using Von Frey filaments and validated questionnaires are lacking. Alloknesis differs from mechanical pruritus in that it is linked to sensitization to pruritus and therefore occurs in pathological conditions, whereas mechanical pruritus (triggered by the presence of insects on the skin, for example) is a physiological phenomenon. While the role of central sensitization to pruritus in alloknesis is still poorly understood, the role of peripheral sensitization is becoming clearer. Interactions between low-threshold mechanoreceptors (LTMRs) and spinal interneurons are especially involved. Both the mechanical labelled pathway and the polymodal pathway have been shown to contribute to mechanical alloknesis. The mechanical labelled pathway comprises dedicated primary sensory neurons, spinal interneurons, and projection neurons that are functionally distinct from those involved in chemical itch. The polymodal pathway relies on a subset of primary sensory neurons traditionally associated with chemical itch, which can also transduce light mechanical stimuli through the activation of the mechanosensitive ion channel PIEZO1. Both converge onto the gastrin-releasing peptide (GRP) - GRP receptor (GRPR) chemical itch pathway in the spinal cord. Alloknesis is largely unknown to healthcare professionals and even more so to patients, and is not actively investigated. The objective of reducing alloknesis should be considered a therapeutic goal. To date, it has not been investigated in clinical trials. A novel research domain is emerging concerning this symptom, which exerts a substantial impact on the daily lives of numerous patients.

Itch is a somatosensory sensation to remove potential harmful stimulation with a scratching desire, which could be divided into mechanical and chemical itch according to diverse stimuli, such as wool fiber and insect biting. It has been reported that neuropeptide Y (NPY) neurons, a population of spinal inhibitory interneurons, could gate the transmission of mechanical itch, with no effect on chemical itch. In our study, we verified that chemogenetic activation of NPY neurons could inhibit the mechanical itch as well as the chemical itch, which also attenuated the alloknesis phenomenon in the chronic dry skin model. Afterwards, intrathecal administration of NPY1R agonist, [Leu31, Pro34]-NPY (LP-NPY), showed the similar inhibition effect on mechanical itch, chemical itch and alloknesis as chemo-activation of NPY neurons. Whereas, intrathecal administration of NPY1R antagonist BIBO 3304 enhanced mechanical itch and reversed the alloknesis phenomenon inhibited by LP-NPY treatment. Moreover, selectively knocking down NPY1R by intrathecal injection of Npy1r siRNA enhanced mechanical and chemical itch behavior as well. These results indicate that NPY neurons in spinal cord regulate mechanical and chemical itch, and alloknesis in dry skin model through NPY1 receptors.

Itch is a sensation in the skin which provokes the desire to scratch. In the past few decades there has been a significant elucidation of the immune and neural pathways which underly the sensation of itch. An interesting divergence in the itch pathway relates to the type of stimulation used to evoke an itchy sensation. Commonly, chemical mediators of itch such as histamine are injected into the skin where they activate their cognate receptors on sensory neurons. Another way to evoke itch, particularly in patients with chronic itch, is to use light mechanical stimulation. Investigation into these pathways utilizing the mouse model have shown that the neuronal pathways which underly chemical itch are distinct from those which mediate itch in response to mechanical stimulation. Specific populations of primary sensory neurons, spinal interneurons and transmission neurons have been identified which suggests a labeled line for itch transmission. Additionally, Piezo channels, which underly mechanosensation, were discovered to play an important role in the mechanical itch pathway. Given these novel findings relating to the mechanical itch pathway, the purpose of this review is to summarize the reports from human subjects and animal studies to highlight the advances in our understanding of mechanical itch and alloknesis.

Identification of an essential spinoparabrachial pathway for mechanical itch

Chronic pruritus is a major unmet clinical problem affecting one in four adults. G protein-coupled receptors (GPCRs) are key receptors driving itch signaling and are a therapeutic target for itch relief. The endosomal signaling of GPCRs provides new challenges for understanding how GPCR signaling is regulated, how endosomal signaling of GPCRs contributes to disease states like chronic pruritus and opens new targets for therapeutic development. The Gastrin releasing peptide receptor (GRPR) is a key mediator of pruritus in the spinal cord. Yet, little is known about the molecular mechanisms regulating GRPR signaling in pruritus, if GRPR can signal from endosomes, or the role of endosomal GRPR in the development of pruritus. Here we show the importance of internalization and endosomal signaling of GRPR in pruritus. Agonist induced GRPR internalization and trafficking was quantified using BRET or microscopy while endosomal-mediated ERK signaling was measured using compartmentalized FRET biosensors. Recruitment of G proteins to endosomes was measured with NanoBit BRET. pH sensitive mesoporous silica nanoparticles (MSN) which accumulated in endosomes were used to deliver RC-3095, a GRPR specific antagonist, intracellularly to block endosomal signaling of GRPR. MSN-RC proved more effective than free RC-3095 at inhibiting chloroquine scratching in mice. Our results demonstrate a critical role for GRPR endosomal signaling in itch sensation. These results highlight the ability of endosomally targeted antagonist to inhibit GRPR signaling and provide a new target for developing therapeutics that block GRPR mediated pruritus.