Hindlimb Scratching Research Articles

A spinal circuit model with asymmetric cervical-lumbar layout controls backward locomotion and scratching in quadrupeds

Locomotion and scratching are basic motor functions which are critically important for animal survival. Although the spinal circuits governing forward locomotion have been extensively investigated, the organization of spinal circuits and neural mechanisms regulating backward locomotion and scratching remain unclear. Here, we extend a model by Danner et al. to propose a spinal circuit model with asymmetrical cervical-lumbar layout to investigate these issues. In the model, the left–right alternation within the cervical and lumbar circuits is mediated by V 0D and V 0V commissural interneurons (CINs), respectively. With different control strategies, the model closely reproduces multiple experimental data of quadrupeds in different motor behaviors. Specifically, under the supraspinal drive, walk and trot are expressed in control condition, half-bound is expressed after deletion of V 0V CINs, and bound is expressed after deletion of V0 (V 0D and V 0V) CINs; in addition, unilateral hindlimb scratching occurs in control condition and synchronous bilateral hindlimb scratching appears after deletion of V 0V CINs. Under the combined drive of afferent feedback and perineal stimulation, different coordination patterns between hindlimbs during BBS (backward-biped-spinal) locomotion are generated. The results suggest that (1) the cervical and lumbar circuits in the spinal network are asymmetrically recruited during particular rhythmic limb movements. (2) Multiple motor behaviors share a single spinal network under the reconfiguration of the spinal network by supraspinal inputs or somatosensory feedback. Our model provides new insights into the organization of motor circuits and neural control of rhythmic limb movements.

Protective effect of pantothenic acid in kainic acid-induced status eilepticus and associated neurodegeneration in mice

Pantothenic acid (PA) is a water-soluble vitamin (Vitamin B) that has recently been investigated in various chemical-induced neurotoxicity studies. The present study was designed to explore the biological importance of PA as a neuromodulator by releasing monoamine oxidase (MAO)-A and MAO-B in kainic acid (KA)-induced status epilepticus and the associated neurodegeneration in mice. The mice were intraperitoneally administered with KA at a dose of 10 mg/kg, and the injection solution was maintained at pH 7.2 ± 0.1 before the injection. Subsequently, the mice were observed for various behavioral changes, such as grooming, rearing, hind limb scratching, urination, defecation, jaw movements, salivation, head nodding, incidence of convulsions, and their latency or any mortality, which were recorded during a 4-h period. Further, the animals were euthanized for biochemical and histopathological analysis. The oxidative stress status was determined by measuring levels of glutathione, superoxide dismutase, nitrites, and catalase enzymes. The MAO-A and MAO-B activities, which represent an indicator of brain memory function, and the level of tumor necrosis factor alpha, which is an inflammatory marker in brain tissues, were also measured. The PA pre-treated mice showed a significant increase in retention with latency, as demonstrated in the passive avoidance test, which indicate its protective effect against the KA-induced cognitive deficit. The results showed that the anti-oxidative and anti-inflammatory potential of PA is due to the change in lipid peroxidation, which may prevent mitochondrial damage in neuronal cell, thereby conferring neuroprotection.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Cinnamaldehyde elicits itch behavior via TRPV1 and TRPV4 but not TRPA1.

Introduction:Cinnamaldehyde (CA) elicits itch sensation in humans. We investigated if CA elicits scratching behavior in mice and determined the roles for TRPV1, TRPA1, and TRPV4.Materials and Methods:Scratching behavior elicited by intradermal injection of CA was assessed in wildtype (WT) mice and knockout (KO) mice lacking TRPV1, TRPA1, TRPV4, or deficient in mast cells. We also assessed scratching and wet dog shakes elicited by low-threshold mechanical stimulation of skin treated topically with CA or vehicle. Using calcium imaging we tested if CA activates dorsal root ganglion (DRG) neurons of each genotype.Results:Intradermal cheek injection of CA elicited dose-dependent hindlimb scratch bouts, with fewer forelimb wipes and facial groom bouts that were not dose-dependent. CA elicited significantly fewer scratch bouts in TRPV1 and TRPV4 KO mice, but not TRPA1KOs, compared with WTs. There were no sex differences across genotypes. The histamine H1 antagonist cetirizine did not affect CA-evoked scratching, which was normal in mast cell deficient mice, indicating lack of histamine involvement. Scores for alloknesis were significantly greater following topical application of CA compared with vehicle. Post-CA alloknesis scores were significantly higher in TRPV4KOs of both sexes and in female TRPV1 and TRPA1KOs, compared with WTs. Low threshold mechanical stimuli also elicited significantly more wet dog shakes in mice treated topically with 20% CA, with significantly fewer in TRPV1, TRPA1, and TRPV4KOs compared with WTs. In calcium imaging studies, CA excited 24% of WT DRG cells, significantly fewer (11.5%) in cells from TRPV4KOs, and none in TRPA1KOs. Responses of cells of all genotypes exhibited significant sensitization to repeated CA stimulation. Sensitization was significantly enhanced by IL-4, which itself excited 16% of WT DRG cells and none from TRPA1KOs.Discussion:The results indicate that TRPA1 is dispensable for CA-evoked scratching, which depends partly on TRPV1 and TRPV4.

Atopic dermatitis induces anxiety- and depressive-like behaviors with concomitant neuronal adaptations in brain reward circuits in mice

Clinically, it has been reported that atopic dermatitis (AD) has been linked with negative emotional problems such as depression and anxiety, thereby reducing the quality of life, but little is known about the molecular mechanism that underlies AD-associated emotional impairments. We sought to determine whether AD could induce anxiety- and depressive-like symptoms in mice and to identify pertinent signaling changes in brain reward circuitry. AD-like lesions were induced by the repeated intradermal application of MC903 into the cheek of the mouse. We assessed dermatitis severity with scratching behavior, histopathological changes, anxiety- and depressive-like behaviors using the elevated plus maze, open field and tail suspension tests, and serum corticosterone levels. In the nucleus accumbens (NAc), dorsal striatum (DS) and ventral tegmental area (VTA), protein levels of dopamine- and plasticity-related signaling molecules were determined by Western immunoblotting assay. Intradermal administration of MC903 into mouse cheek provoked a strong hind limb scratching behavior as well as the robust skin inflammation with epidermal thickening. MC903-treated mice also displayed markedly increased anxiety- and depressive-like behaviors, along with elevated serum corticosterone levels. Under these conditions, enhanced cAMP response element binding protein (CREB) and dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) phosphorylation, significantly higher brain-derived neurotrophic factor (BDNF) and ΔFosB, but reduced tyrosine hydroxylase (TH) and dopamine D1 receptor (D1R) protein expression were found in the NAc, DS and VTA. Striatal BDNF, phospho-DARPP32 and phospho-CREB levels were significantly associated with the levels of depressive-like behavior in these mice. Taken together, these findings demonstrate that AD-like skin lesion elicits anxiety- and depressive-like phenotypes that are associated with neuroplasticity-related changes in reward circuitry, providing a better understanding of AD-associated emotional impairments.

Aimed limb movements in a hemimetabolous insect are intrinsically compensated for allometric wing growth by developmental mechanisms.

ABSTRACTFor aimed limb movements to remain functional, they must be adapted to developmental changes in body morphology and sensory-motor systems. Insects use their limbs to groom the body surface or to dislodge external stimuli, but they face the particular problem of adapting these movements to step-like changes in body morphology during metamorphosis or moulting. Locusts are hemimetabolous insects in which the imaginal moult to adulthood results in a sudden and dramatic allometric growth of the wings relative to the body and the legs. We show that, despite this, hind limb scratches aimed at mechanosensory stimuli on the wings remain targeted to appropriate locations after moulting. In juveniles, the tips of the wings extend less than halfway along the abdomen, but in adults they extend well beyond the posterior end. Kinematic analyses were used to examine the scratching responses of juveniles (fifth instars) and adults to touch of anterior (wing base) and posterior (distal abdomen) targets that develop isometrically, and to wing tip targets that are anterior in juveniles but posterior in adults. Juveniles reach the (anterior) wing tip with the distal tibia of the hind leg using anterior rotation of the thoraco-coxal and coxo-trochanteral (‘hip’) joints and flexion of the femoro-tibial (‘knee’) joint. Adults, however, reach the corresponding (but now posterior) wing tip using posterior rotation of the hip and extension of the knee, reflecting a different underlying motor pattern. This change in kinematics occurs immediately after the adult moult without learning, indicating that the switch is developmentally programmed.

LB1006 Mouse model of post-burn itch

The majority of the post-burn patients suffer from chronic itch, which is often resistant to antihistamine treatment. Post-burn itch involves sensitization of itch-signaling pathways, leading to ongoing itch and alloknesis (touch-evoked itch), but the underlying mechanisms behind post-burn itch are largely unknown. We have developed a novel animal model to investigate the neuronal mechanisms of post-burn itch. A scald burn injury was produced in adult C57BL/6 mice by exposing the shaved back skin to boiling water. The burn surface area was over 150 mm2 on Day 1, decreased to 30 mm2 on Day 14, and then remained at a plateau. To assess spontaneous scratching, mice were videotaped on Days 0, 1, 3, 5, 7, 10, 14, 21, and 28 after the scald burn treatment. Counts of spontaneous scratch bouts increased transiently on Days 1, 3, and Day 5 compared to Day 0, returned to the basal level by Day 10, and reincreased significantly on Days 14, 21, and 28. To test for alloknesis, a weak von Frey filament (VF; 0.7 mN) was repeatedly applied to post-burn skin on Days 0, 1, 3, 5, 7, 14, 21, and 28 after the scald burn treatment, and the presence or absence of evoked hindlimb scratch bouts was noted (VF stimulation does not elicit any response in naïve C57BL/6 mice). VF-evoked scratching increased significantly on Days 21 and 28. The histamine H1 receptor antagonist chlorpheniramine was tested on Day 22 but did not inhibit spontaneous scratching, suggesting that non-histaminergic itch pathways are involved in post-burn itch. This new animal model appears to be useful for investigations of post-burn itch and sensitization of itch-signaling pathways.

Spinal Cord Preparation from Adult Red-eared Turtles for Electrophysiological Recordings during Motor Activity.

Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to anoxia allows for investigation of the fully developed and adult spinal circuitry, as opposed to mammals, which are sensitive to anoxia and where using neonates are often required to remedy the problems. The turtle is mechanically stable and natural sensory inputs can induce multiple complex motor behaviors, without the need for application of neurochemicals. Here, we provide a detailed protocol of how to make the adult turtle preparation, also known as the integrated preparation for electrophysiological investigation. Here, the hind-limb scratch reflex can be induced by mechanical sensory activation, while recording single cells, and the network activity, via intracellular-, extracellular- and electroneurogram recordings. The preparation was developed for the studies by Petersen et al. (2014) and Petersen and Berg (2016), and other ongoing studies.

Role of ERK1/2 activation on itch sensation induced by bradykinin B1 activation in inflamed skin.

It has previously been demonstrated that bradykinin receptor B1 (B1R) agonists evoke an itch-related scratching response in inflamed skin via the B1 receptor; however, the mechanisms responsible for this abnormal itch sensation remain unclear. Therefore, the present study utilized a complete Freund's adjuvant (CFA)-induced mouse model of inflammation to elucidate the mechanisms responsible. Over a period of 30 min, scratching behavior was quantified by the number of hind limb scratches of the area surrounding the drug injection site on the neck. Furthermore, western blot analysis was used to investigate the potential role of extracellular signal-regulated kinase (ERK) 1/2 signaling as a mediator of itch in CFA-treated mice. The results demonstrated that CFA-induced inflammation at the back of the neck is associated with sustained enhancement of ERK1/2 activation in the spinal cord. Moreover, B1R agonist treatment resulted in increased expression of phosphorylated ERK1/2 in the spinal cord, which peaked at 45 min. Consistent with these findings, inhibition of either mitogen-activated protein/ERK kinase or ERK1/2, as well as inhibition of ERK1/2 activation following inflammation, attenuated B1 receptor-mediated scratching responses to a greater extent, as compared with control mice. Collectively, the results of the present study indicated that enhanced and persistent ERK1/2 activation in the spinal cord may be required to induce a scratching response to B1R agonists following CFA-induced inflammation.

Adrenergic β2-receptor mediates itch hypersensitivity following heterotypic chronic stress in rats.

Chronic stress is widely considered to trigger or enhance itch, especially for pruritic dermatitis. However, the molecular mechanisms linking chronic stress and itch are still unknown. The present study aimed to elucidate the role of adrenergic signaling in itch hypersensitivity following heterotypic chronic intermittent stress (HIS) in rats. HIS significantly increased hindlimb scratching, but not forepaw swiping, induced by intradermal injection of 5-hydroxytryptamine (5-HT) in the rat cheek. Coadministration of stress mediators such as norepinephrine or epinephrine dose-dependently increased both 5-HT-induced hindlimb scratching and 5-HT-induced forepaw swiping. HIS-induced itch hypersensitivity was attenuated by blockade of sympathetic signaling through guanethidine treatment, and systemic administration of the β-adrenoceptor antagonist propranolol and the β2-adrenoceptor antagonist butoxamine, but not on treatment with an α-adrenoceptor antagonist phentolamine and a β1-adrenoceptor antagonist atenolol. Moreover, HIS selectively increased the expression of β2-adrenoceptors and proinflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nerve growth factor (NGF)] in rat skin. The β-blockers propranolol and butoxamine abolished the upregulation of proinflammatory factors. The β2-adrenoceptor agonist terbutaline was sufficient to enhance the skin expression of TNF-α and IL-1β and to increase 5-HT-induced scratching in naive rats. Pretreatment with TNF-α could increase 5-HT-induced scratching. Together, these results demonstrate that β2-adrenoceptors mediate itch hypersensitivity following chronic stress by inducing proinflammatory factors, such as TNF-α, in the skin.

Intrathecal morphine-3-glucuronide-induced nociceptive behavior via Delta-2 opioid receptors in the spinal cord

Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces severe hindlimb scratching followed by biting and licking in mice. The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with an antisera against dynorphin. However, the selective κ-opioid receptor antagonist, nor-BNI did not prevent the M3G-induced behavioral response. Dynorphin is rapidly degraded by a dynorphin-converting enzyme (cystein protease), to leucine-enkephalin (Leu-ENK). The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with the antisera against Leu-ENK. We also showed that M3G co-administered with Leu-ENK-converting enzyme inhibitors, phosphoramidon and bestatin produced much stronger behavioral responses than M3G alone. Furthermore, the M3G-induced behavioral responses were inhibited dose-dependently by i.t. co-administration of the non-selective δ-opioid receptor antagonist, naltrindole or the selective δ2-opioid receptor antagonist, naltriben, whereas the selective δ1-opioid receptor antagonist, BNTX had no effect. An i.t. injection of M3G also produced a definite activation of ERK in the lumbar dorsal spinal cord. Western blotting analysis revealed that antisera against dynorphin, antisera against Leu-ENK, naltrindole or naltriben resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that M3G-induced nociceptive responses and ERK activation may be triggered via δ2-opioid receptors activated by Leu-ENK, which is formed from dynorphin in the spinal cord.

Divisive gain modulation of motoneurons by inhibition optimizes muscular control.

When using muscles, the precision with which force is delivered is as important as the delivery of force itself. Force is regulated by both the number of recruited motoneurons and their spike frequency. While it is known that the recruitment is ordered to reduce variability in force, it remains unclear whether the motoneuron gain, i.e., the slope of the transformation between synaptic input and spiking output, is also modulated to reduce variability in force. To address this issue, we use turtle hindlimb scratching as a model for fine motor control, since this behavior involves precise limb movement to rub the location of somatic nuisance touch. We recorded intracellularly from motoneurons in a reduced preparation where the limbs were removed to increase mechanical stability and the motor nerve activity served as a surrogate for muscle force. We found that not only is the gain of motoneurons regulated on a subsecond timescale, it is also adjusted to minimize variability. The modulation is likely achieved via an expansive nonlinearity between spike rate and membrane potential with inhibition having a divisive influence. These findings reveal a versatile mechanism of modulating neuronal sensitivity and suggest that such modulation is fundamentally linked to optimization.

Nalfurafine Suppresses Pruritogen- and Touch-evoked Scratching Behavior in Models of Acute and Chronic Itch in Mice

The kappa-opioid agonist, nalfurafine, has been approved in Japan for treatment of itch in patients with chronic kidney disease. We presently investigated if systemic administration of nalfurafine inhibited ongoing or touch-evoked scratching behavior (alloknesis) following acute intradermal injection of histamine or the non-histaminergic itch mediator, chloroquine, in mice. We also investigated if nalfurafine suppressed spontaneous or touch-evoked scratching in an experimental model of chronic dry skin itch. Nalfurafine reduced scratching evoked by histamine and chloroquine. Following acute histamine, but not chloroquine, low-threshold mechanical stimuli reliably elicited directed hindlimb scratching behavior, which was significantly attenuated by nalfurafine. In mice with experimental dry skin, nalfurafine abolished spontaneous scratching but had no effect on alloknesis. Nalfurafine thus appears to be a promising treatment for acute itch as well as ongoing itch of dry skin.

Dense distributed processing in a hindlimb scratch motor network.

In reduced preparations, hindlimb movements can be generated by a minimal network of neurons in the limb innervating spinal segments. The network of neurons that generates real movements is less well delineated. In an ex vivo carapace-spinal cord preparation from adult turtles (Trachemys scripta elegans), we show that ventral horn interneurons in mid-thoracic spinal segments are functionally integrated in the hindlimb scratch network. First, mid-thoracic interneurons receive intense synaptic input during scratching and behave like neurons in the hindlimb enlargement. Second, some mid-thoracic interneurons activated during scratching project descending axons toward the hindlimb enlargement. Third, elimination of mid-thoracic segments leads to a weakening of scratch rhythmicity. We conclude that densely innervated interneurons in mid-thoracic segments contribute to hindlimb scratching and may be part of a distributed motor network that secures motor coherence.

Spinal ERK2 activation through δ2-opioid receptors contributes to nociceptive behavior induced by intrathecal injection of leucine-enkephalin

Intrathecal (i.t.) injection of leucine-enkephalin (Leu-ENK), co-administered with peptidase inhibitors, phosphoramidon (an endopeptidase 24.11 inhibitor), and bestatin (a general aminopeptidase inhibitor), produced behaviors consisting of the biting and/or licking of the hindpaw and the tail along with hindlimb scratching directed toward the flank, which peaked at 10–15min after an injection. This characteristic behavior was not observed in mice treated with i.t. Leu-ENK alone. We also investigated the effect of the extracellular signal-regulated kinase (ERK) in spinal processing of nociception induced by i.t. co-administration of Leu-ENK with phospharamidon and bestatin. Western blot analysis of phospho-ERK (pERK) showed a significant increase of pERK2 in the lumbar spinal cord in response to i.t. Leu-ENK co-injected with peptidase inhibitors. The MAP kinase-ERK inhibitor, U0126 dose-dependently attenuated the nociceptive behavior and spinal ERK activation to i.t. Leu-ENK co-injected with peptidase inhibitors. Furthermore, the nociceptive behavior and spinal ERK activation evoked by i.t. Leu-ENK in combination with peptidase inhibitors were inhibited by co-administration of the non-selective δ-opioid receptor antagonist, naltrindole, the selective δ2-opioid receptor antagonist, naltriben, the non-competitive N-methyl-d-aspartate (NMDA) antagonist, MK-801 or the non-selective nitric oxide synthase inhibitor, l-NAME, the selective nNOS inhibitor, Nω-propyl-l-arginine or the selective iNOS inhibitor, W1400, but not by the selective δ1-receptor antagonist, BNTX (7-benzylidenenaltrexone). These results suggest that spontaneous nociceptive behaviors produced by i.t. co-administration of Leu-ENK with peptidase inhibitors may be induced by an activation of the glutamate-NO-ERK pathway through the δ2-opioid receptor in the dorsal spinal cord.

Angiotensin II Produces Nociceptive Behavior through Spinal AT1 Receptor-Mediated p38 Mitogen-Activated Protein Kinase Activation in Mice

BackgroundIt has been demonstrated that angiotensin II (Ang II) participates in either the inhibition or the facilitation of nociceptive transmission depending on the brain area. Neuronal Ang II is locally synthesized not only in the brain, but also in the spinal cord. Though the spinal cord is an important area for the modulation of nociception, the role of spinal Ang II in nociceptive transmission remains unclear. Therefore, in order to elucidate the role of Ang II in nociceptive transmission in the spinal cord, we examined the effect of intrathecal (i.t.) administration of Ang II into mice.ResultsI.t. administration of Ang II produced a behavioral response in mice mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by Ang II (3 pmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.1-0.3 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also inhibited dose-dependently by i.t. co-administration of losartan (0.3-3 nmol), an Ang II type 1 (AT1) receptor antagonist, and SB203580 (0.1-1 nmol), a p38 MAPK inhibitor. However, the Ang II type 2 (AT2) receptor antagonist PD123319, the upstream inhibitor of ERK1/2 phosphorylation U0126, and the JNK inhibitor SP600125 had no effect on Ang II-induced nociceptive behavior. Western blot analysis showed that the i.t. injection of Ang II induced phosphorylation of p38 MAPK in the lumbar dorsal spinal cord, which was inhibited by losartan, without affecting ERK1/2 and JNK. Furthermore, we found that AT1 receptor expression was relatively high in the lumbar superficial dorsal horn.ConclusionsOur data show that i.t. administration of Ang II induces nociceptive behavior accompanied by the activation of p38 MAPK signaling mediated through AT1 receptors. This observation indicates that Ang II may act as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information.

Effects of acute stressors on itch- and pain-related behaviors in rats

Many acute stressors reduce pain, a phenomenon called stress-induced antinociception (SIA). Stress also is associated with increased scratching in chronic itch conditions. We investigated effects of acute stressors on facial itch and pain using a recently introduced rat model. Under baseline (no-swim) conditions, intradermal (id) cheek microinjection of the pruritogen serotonin (5-HT) selectively elicited hindlimb scratch bouts, whereas the algogen mustard oil (allyl isothiocyanate [AITC]) selectively elicited ipsilateral forepaw swipes, directed to the cheek injection site. To test effects of swim stress, rats received id cheek microinjection of 5-HT (1%), AITC (10%), or vehicle, and were then subjected to one of the following swim conditions: (1) weak SIA (W-SIA), (2) naltrexone-sensitive SIA (intermediate or I-SIA), or (3) naltrexone-insensitive SIA (strong or S-SIA). After the swim, we recorded the number of hindlimb scratch bouts and forelimb swipes directed to the cheek injection site, as well as facial grooming by both forepaws. Under S-SIA, AITC-evoked swiping and 5-HT–evoked scratching were both reduced. I-SIA reduced AITC-evoked swiping with no effect on 5-HT–evoked scratching. Facial grooming immediately post-swim was suppressed by S-SIA, but not I- or W-SIA. W-SIA tended to equalize scratching and swiping elicited by 5-HT and AITC compared with no-swim controls, suggesting altered itch and pain processing. Exercise (wheel-running), novelty, cold exposure, and fear (shaker table), key components of swim stress, differentially affected tail-flick latencies and 5-HT–evoked swiping and scratching behavior. Thus, itch and pain can be simultaneously suppressed by a combination of acute stress-related factors via an opioid-independent mechanism.

Mouse Model of Touch-Evoked Itch (Alloknesis)

Lightly touching normal skin near a site of itch can elicit itch sensation, a phenomenon known as alloknesis. To investigate the neural mechanisms of alloknesis, we have developed an animal model. Low-threshold mechanical stimulation of the skin normally does not elicit any response in naïve C57/BL6 mice. Following acute intradermal (id) injection of histamine in the rostral back, mechanical stimulation 7 mm from the injection site elicited discrete hindlimb scratch bouts directed toward the stimulus. This began at 10 min and peaked 20–40 min post-histamine, declining over the next hour. Histamine itself elicited bouts of scratching not associated with the mechanical stimulus, that ceased after 30 min. Histamine- and touch-evoked scratching was inhibited by the μ-opiate antagonist naltrexone. Touch-evoked scratching was observed following id 5-HT, a PAR-4 agonist and a MrgprC11 agonist BAM8-22, but not chloroquine or a PAR-2 agonist. The histamine H1 receptor antagonist terfenadine prevented scratching and alloknesis evoked by histamine, but not 5-HT, a PAR-4 agonist or a MrgprC11 agonist. In mice with experimental dry skin, there was a time-dependent increase in spontaneous and touch-evoked scratching. This animal model, which to our knowledge is previously unreported, appears to be useful to investigate neural mechanisms of itch and alloknesis.

Differences in peripheral endocannabinoid modulation of scratching behavior in facial vs. spinally-innervated skin

Cannabinoids suppress nocifensive behaviors in rodents. We presently investigated peripheral endocannabinoid modulation of itch- and pain-related behaviors elicited from facial vs. spinally-innervated skin of rats. Intradermal (id) injection of the pruritogen serotonin (5-HT) elicited significantly more hindlimb scratch bouts, and longer cumulative time scratching, when injected in the rostral back compared to the cheek. Pretreatment of skin with inhibitors of degrading enzymes for the endocannabinoids anandamide (URB597) or 2-arachidonoylglycerol (JZL184) significantly reduced scratching elicited by 5-HT in the rostral back. These effects were prevented by co-treatment with antagonists of the CB1 (AM251) or CB2 receptor (AM630), implicating both receptor subtypes in endocannabinoid suppression of scratching in spinally-innervated skin. Conversely, pretreatment with either enzyme inhibitor, or with AM630 alone, increased the number of scratch bouts elicited by id 5-HT injection in the cheek. Moreover, pretreatment with JZL184 also significantly increased pain-related forelimb wipes directed to the cheek following id injection of the algogen, allyl isothiocyanate (AITC; mustard oil). Thus, peripheral endocannabinoids have opposite effects on itch-related scratching behaviors in trigeminally- vs. spinally-innervated skin. These results suggest that increasing peripheral endocannabinoid levels represents a promising therapeutic approach to treat itch arising from the lower body, but caution that such treatment may not relieve, and may even exacerbate, itch and pain arising from trigeminally-innervated skin of the face or scalp.

Non-opioid nociceptive activity of human dynorphin mutants that cause neurodegenerative disorder spinocerebellar ataxia type 23

We previously identified four missense mutations in the prodynorphin gene that cause human neurodegenerative disorder spinocerebellar ataxia type 23 (SCA23). Three mutations substitute Leu5, Arg6, and Arg9 to Ser (L5S), Trp (R6W) and Cys (R9C) in dynorphin A(1–17) (Dyn A), a peptide with both opioid activities and non-opioid neurodegenerative actions. It has been reported that Dyn A administered intrathecally (i.t.) in femtomolar doses into mice produces nociceptive behaviors consisting of hindlimb scratching along with biting and licking of the hindpaw and tail (SBL responses) through a non-opioid mechanism. We here evaluated the potential of the three mutant peptides to produce similar behaviors. Compared to the wild type (WT)-peptide, the relative potency of Dyn A R6W, L5S and R9C peptides for SBL responses was 50-, 33- and 2-fold higher, and Dyn A R6W and L5S induced the SBL responses at a 10–30-fold lower doses. Dyn A R6W was the most potent peptide. The SBL responses induced by Dyn A R6W were dose dependently inhibited by morphine (i.p.; 0.1–1mg/kg) or MK-801, an NMDA ion channel blocker (i.t. co-administration; 5–7.5nmol). CP-99,994, a tachykinin NK1 receptor antagonist (i.t. co-administration; 2nmol) and naloxone (i.p.; 5mg/kg) failed to block effects of Dyn A R6W. Thus, similarly to Dyn A WT, the SBL responses induced by Dyn A R6W may involve the NMDA receptor but are not mediated through the opioid and tachykinin NK1 receptors. Enhanced non-opioid excitatory activities of Dyn A mutants may underlie in part development of SCA23.