Intrinsic braking role of descending locus coeruleus noradrenergic neurons in acute and chronic itch in mice

Itch is defined as an unpleasant sensation that provokes a desire to scratch. Our understanding of neuronal circuits for itch information transmission and processing in the spinal dorsal horn (SDH) has progressively advanced following the identification of SDH neuron subsets that are crucial for scratching behavior in models of itch. However, little is known about the control of acute and chronic itch by descending signals from the brain to the SDH. In this study, using genetic approaches that enable cell-type and circuit-specific functional manipulation, we reveal an intrinsic potential of locus coeruleus (LC)-noradrenergic (NAergic) neurons that project to the SDH to control acute and chronic itch. Activation and silencing of SDH-projecting LC-NAergic neurons reduced and enhanced scratching behavior, respectively, in models of histamine-dependent and -independent acute itch. Furthermore, in a model of chronic itch associated with contact dermatitis, repetitive scratching behavior was suppressed by the activation of the descending LC-NAergic pathway and by knocking out NA transporters specific to descending LC-NAergic neurons using a CRISPR-Cas9 system. Moreover, patch-clamp recording using spinal slices showed that noradrenaline facilitated inhibitory synaptic inputs onto gastrin-releasing peptide receptor-expressing SDH neurons, a neuronal subset known to be essential for itch transmission. Our findings suggest that descending LC-NAergic signaling intrinsically controls acute and chronic itch and provide potential therapeutic strategies for the treatment of acute and chronic itch.


Introduction
Itch is defined as an unpleasant cutaneous sensation that provokes the desire to scratch, and scratching can transiently relieve such itching sensations [1]. However, in pathological conditions such as atopic and contact dermatitis, itch sensation becomes intense and chronic, which leads to excessive, repetitive scratching. As the existing treatments (e.g., antihistamines) are largely ineffective, the elucidation of the mechanisms underlying chronic itch and the development of novel therapeutic agents are crucial.
Pruriceptive information is conveyed via primary afferents from the skin and processed in the spinal dorsal horn (SDH). Recent studies have progressively advanced our understanding of the mechanism underlying neuronal circuits for itch transmission in the nervous system [2][3][4][5]. Specifically, gastrin-releasing peptide receptor (GRPR)-expressing (GRPR + ) neurons in the SDH act as a hub for converging pruriceptive information and are essential for producing scratching behaviors in diverse models of acute and chronic itch [6][7][8]. Furthermore, similar to the regulation of nociceptive transmission, pruriceptive transmission in the SDH has been considered to also be remotely controlled by the brain through descending neuronal pathways. Noradrenaline (NA) and serotonin (5-HT) are the two major monoamines that are utilized as neurotransmitters in the descending pathways. A recent study has shown that scratching behavior is suppressed in mice with decreased spinal 5-HTergic terminals and lacking the enzyme for 5-HT synthesis [9]. Activation of 5-HT1A receptors potentiates the excitation of GRPR + neurons via the enhancement of gastrinreleasing peptide (GRP)-induced responses. This suggests that the activation of descending 5-HTergic pathways facilitates itch transmission via GRPR signaling in the SDH [9]. On the other hand, intrathecal injection of agonists for α 1 -or α 2 -adrenaline receptors (ARs) has been shown to inhibit acute itch-related scratching behavior [10]. It has also been reported that antidepressants that can increase spinal NA and/or 5-HT levels reduce scratching in mouse models of chronic itch [11] and in humans [12,13], suggesting that endogenous spinal NA plays an inhibitory role in itching. Supporting this, there is an inverse correlation between itch-related scratching behavior and spinal NA content [10]. The locus coeruleus (LC) is the major brain region that contains SDHprojecting NAergic cell bodies [14,15]. While intrathecal treatment with 6-hydroxydopamine (6-OHDA), a neurotoxin that can cause the degeneration of catecholaminergic neurons, has been reported to exacerbate scratching behavior [10], there is no direct evidence for the role of SDH-projecting LC-NAergic neurons in controlling acute and chronic itch.
In this study, using genetic tools that enable cell-type and circuit-specific functional manipulation, we demonstrated for the first time the potential ability of SDHprojecting LC-NAergic neurons to regulate scratching behavior in models of acute and chronic itch and also to enable the development of drugs to relieve itch sensation.

NA facilitates inhibitory synaptic inputs on itch-transmission neurons via α 1A -ARs
Behavioral data obtained in our study suggest that spinal NA would have an inhibitory effect on itch neurotransmission in the SDH. Our previous findings showed that activation of SDH inhibitory interneurons powerfully  Fig. 1a. The time course of scratching behavior (a: n = 7, *P < 0.05) and the summary of total scratching behavior (b: n = 7, ***P < 0.001). Behavior was measured for 5 h after saline or CNO (10 mg/kg, i.p.) administration. Data show the mean ± SEM suppresses chronic itch [25], and GRPR + neurons have been shown to be controlled by local inhibitory interneurons [26,27]. Thus, we predicted that NA modulates inhibitory synaptic inputs onto GRPR + neurons. To examine this, we performed patch-clamp recording from GRPR + neurons using spinal cord slices from Grpr-EGFP mice [28,29] and measured inhibitory postsynaptic currents (IPSCs) (Fig. 5a). Spontaneous IPSCs of GRPR + neurons were markedly facilitated by the application of NA (Fig. 5b). The frequency of IPSCs was significantly increased by NA, while the average amplitude of IPSCs was not changed (Fig. 5d). A similar facilitation of IPSC frequency in GRPR + neurons was observed after the application of the α 1 -AR agonist phenylephrine or the α 1A -AR agonist A61603 [30] (Fig. 5c, d). These data suggest that spinal NA facilitates inhibitory synaptic inputs on GRPR + neurons in the SDH presumably via α 1A -ARs.

Discussion
By using genetic approaches that enable cell-type and circuit-specific functional manipulation, we demonstrate for the first time that SDH-projecting LC-NAergic neurons powerfully control itch-related behavior. Indeed, stimulation of this pathway markedly suppressed scratching in models of histamine-dependent and -independent acute itch. Conversely, silencing the descending LC-NAergic pathway increased these itching behavioral responses. Thus, descending LC-NAergic neurons have an intrinsic ability to suppress acute itch. This hypothesis is supported by previous reports that intrathecally injected catecholaminergic neurotoxin 6-OHDA or α-AR antagonists increases scratching in acute itch models [10]. Furthermore, a marked suppression of scratching in mice with contact dermatitis was observed during acute stimulation of descending LC-NAergic neurons. This suggests that the inhibitory action of this pathway persists even under chronic itch conditions. The intrinsic potential of descending LC-NAergic neurons to suppress chronic itch was demonstrated by subsequent experiments, which showed that knocking out NET specifically in SDH-projecting LC-NAergic neurons using the CRISPR-Cas9 system resulted in the suppression of scratching in the DCP model. Consistent with our findings, NA and/or 5-HT reuptake inhibitors have also been shown to ameliorate chronic itch in mice [11] and in humans [12,13]. Our findings, together with the findings of the aforementioned studies, suggest that the endogenous spinal NA released from descending LC-NAergic neurons may have an inhibitory effect on itch transmission in the SDH, although whether scratching behavior caused by other pruritogens is also modulated by descending LC-NAergic neurons is an important subject.
Given that intrathecal phenylephrine inhibits scratching [10], spinal α 1 -ARs could be responsible for the antipruritic effect of spinal NA. In the SDH, α 1 -ARs are preferentially expressed in inhibitory interneurons [31], and NA facilitates inhibitory synaptic inputs in the substantia gelatinosa [32]. GRPR + SDH neurons have also been reported to form synaptic connections with inhibitory interneurons [26,27]; however, whether NA facilitates inhibitory inputs to GRPR + neurons was previously unknown. In our study, we demonstrated that NA facilitates inhibitory transmission in GRPR + neurons. A similar facilitation was observed after α 1A -AR agonist application, suggesting a role of α 1A -ARs. The absence of change in the amplitude of IPSCs following NA release implies that NA acts on inhibitory interneurons that project onto GRPR + neurons rather than acting directly on GRPR + neurons. This is supported by recent studies that showed that the level of α 1A -AR mRNA is high in galanin + or preprodynorphin + spinal interneurons [33], which are subsets of inhibitory interneurons that inhibit GRPR + neurons [26,27], but is low in GRPR + neurons [31]. Thus, it is postulated that spinal NA derived from descending LC-NAergic neurons may activate these inhibitory interneurons via α 1A -ARs, inhibit GRPR + neurons, and suppress acute and chronic itch. However, from the data showing that inhibition of preprodynorphin + inhibitory neurons by somatostatin causes spontaneous scratching [34] but chemogenetic inhibition of descending LC-NAergic neurons did not, it appears unlikely that LC-NAergic signals potently maintain basal activity of SDH inhibitory interneurons with a high level under normal conditions without any pruritic signals. Thus, LC-NAergic neurons could be activated in response to pruritic signals and produce their suppressive effect on scratching behavior. In addition to α 1A -ARs, α 2 -ARs could also play a role in regulating itch transmission. Indeed, α 2 -ARs are expressed in primary afferent pruriceptors that express either Mas-related G-protein coupled receptor member A3 or natriuretic peptides B [35], and also in GRPR + SDH neurons [31]. Activating α 2 -ARs at presynaptic terminals of primary afferents reduces excitatory neurotransmission [36]. Intrathecal administration of α 2 -AR agonists inhibits acute itch responses [10,37]. Therefore, the inhibitory effect of descending LC-NAergic neurons on acute and chronic itch could be the result of the combined effect of α 1A -and α 2 -ARs signals. In addition, almost all neurons in the LC are known to produce NA [38], but subsets of LC neurons have also been shown to express some neuropeptides [39]. Additional experiments would be required to determine the role of neuropeptides in LC-NAergic LC-NAergic neurons also send ascending projections throughout the brain that modulate several key brain functions [38,40]. Recently, it was reported that the activation of LC-NAergic neurons that project to the anterior cingulate cortex induces scratching behaviors [41]. Thus, it is possible that ascending and descending LC-NAergic neurons play a distinct role in the control of itch behavior. However, how their neuronal activity is differentially regulated under acute and chronic itch conditions remains unclear.
In summary, we showed that SDH-projecting LC-NAergic neurons have an intrinsic potential to suppress acute and chronic itch and that NA facilitates inhibitory synaptic inputs onto GRPR + SDH neurons presumably via α 1A -ARs. Our finding that descending LC-NAergic neuron-specific NET knockout exerts an ameliorating effect on chronic itch and previous data showing that NA and/or 5-HT reuptake inhibitors reduce chronic itch in mouse models [42], including the DCP model [11], and in humans [12,13], indicate that descending LC-NAergic signaling could be targeted to treat chronic itch.

Animals
C57BL/6J mice (Jackson Laboratory, USA), Grpr-EGFP mice (STOCK Tg(Grpr-EGFP) PZ62Gsat/Mmucd ) (MMRRC, USA) [28] and Dbh-Cre mice (B6.Cg-Tg(B6.Cg-Tg(DBHcre) 9-9Koba/KobaRbrc ) (RIKEN BRC, Japan) [19] were used. All mice used were male and 8-12 weeks of age at the start of each experiment and were housed at 22 ± 1 °C with a 12-h light-dark cycle. All animals were fed food and water ad libitum. All animal experiments were conducted according to relevant national and international guidelines contained in the ' Act on Welfare and Management of Animals' (Ministry of Environment of Japan) and 'Regulation of Laboratory Animals' (Kyushu University) and under the protocols approved by the Institutional Animal Care and Use committee review panels at Kyushu University.

Immunohistochemistry
Immunohistochemical experiments were performed according to the methods in our previous study [22]. Mice were deeply anesthetized by i.p. injection of pentobarbital and perfused transcardially with phosphate buffered saline (PBS), followed by ice-cold 4% paraformaldehyde (PFA)/PBS. The brains were removed, postfixed in the same fixative for overnight at 4 °C and placed in 30% sucrose solution for two overnight at 4 °C. Transverse brain sections (40 μm) were made and immunostained. Primary and secondary antibodies used were Immunofluorescence images were obtained with a confocal laser microscope (LSM700, Carl Zeiss, Germany). Fluorescent intensity of TH or NET was quantified using Fiji (https ://fiji.sc).

Mouse models of acute and chronic itch
Behavioral tests using models of acute and chronic itch was performed by the methods in our previous studies [22,29]. For acute itch models, mice were shaved on the back until one day before injection. CNO (10 mg kg −1 , Enzo Life Science, USA) or varenicline (0.5 mg kg −1 ; Tocris Bioscience) were dissolved in saline and intraperitoneally administered 20 min before intradermal injection of pruritogens [chloroquine (200 µg/50 µl; C6628, Sigma) and compound 48/80 (50 µg/50 µl; C2313, Sigma)] into the shaved back. After the injection, the mouse was placed in a plastic chamber (11 cm in diameter, 10 cm high). Hind limb scratching behavior directed toward the injection site was observed for 30 min. One scratch was defined as a lifting of the hind limb toward the injection site and then placing the limb back on the floor, regardless of how many scratching strokes took place between those two movements [6]. For the basal spontaneous scratching behavior analysis, we counted the scratching behaviors for 15 min after intraperitoneally administration of CNO, varenicline or saline. For a chronic itch model of contact dermatitis, mice were shaved on the back and topically applied by painting 0.2 ml of 1% diphenylcyclopropenone (DCP; Wako) dissolved in acetone under isoflurane anesthesia. Seven days after the first painting (day 7), DCP was painted again on the same area of skin. Seven days later (day 14), measurement of scratching behavior and other experiments [immunohistochemistry or transepidermal water loss (TEWL: see below)] were performed. Scratching behavior in mice was automatically detected and objectively evaluated using MicroAct (Neuroscience, Japan) in accordance with a method described previously [22]. Under isoflurane anesthesia, a small Teflon-coated magnet (1 mm in diameter, 3 mm in length, Neuroscience) was implanted subcutaneously into the hindpaws of the mice at least 1 day before the first recording. Each mouse with implanted magnet was placed in an observation chamber (11 cm in diameter, 18 cm high) with food and tap water, surrounded by a round coil. Movement of magnets implanted subcutaneously into the hindpaws induced electric currents in the coil, which were amplified and recorded by MicroAct software. The analysis parameters for detecting scratch movements were as follows: threshold, 0.07 V; event gap, 0.2 s; minimum duration, 0.2 s; maximum frequency, 35 Hz; minimum frequency, 2 Hz; minimum beats, 2. Scratching behavior was shown as the number of total scratching responses over 5 or 24 h.

Measurement of transepidermal water loss
Transepidermal water loss (TEWL) was measured using the Tewameter TM300 system and a multi-probe adaptor (CK electronic, Germany), in accordance with manufacturer instructions and our previous studies [29]. Under isoflurane anesthesia, the probe collar was placed on the surface of the skin on the animal's back for 20-30 s. Measurements were obtained twice for the left and right sides of the skin, and the values were averaged.

Evaluation of dermatitis
Severity of dermatitis of the face, ears, and the rostral part of the body was assessed as previously described [22,46]: no symptoms (score 0), mild (score 1), moderate (score 2), and severe (score 3). This scoring system was separately applied to the severity of erythema/hemorrhage, edema, excoriation/erosion, and scaling/dryness. The total score (minimum 0, maximum 12) was expressed as the sum of each score of the above four symptoms.