Animal Studies

The relaxin peptide signaling system is involved in diverse physiological processes, but its possible roles in the brain, including nociception, are largely unexplored.

The Acid Sensing Ion Channel 3 (ASIC3) is a non-selective cation channel that is activated by acidification, and is known to have a role in regulating inflammatory pain. It has pro-algesic roles in a range of conditions that present with bone pain, but the mechanism for this has not yet been demonstrated. We aimed to determine if ASIC3 is expressed in Aδ and/or C fiber bone afferent neurons, and to explore its role in the activation and sensitization of bone afferent neurons after acute inflammation. A combination of retrograde tracing and immunohistochemistry was used to determine expression of ASIC3 in the soma of bone afferent neurons. A novel, electrophysiological bone-nerve preparation was used to make recordings of the activity and sensitivity of bone afferent neurons in the presence of carrageenan-induced inflammation, with and without the selective ASIC3 inhibitor APET×2. A substantial proportion of bone afferent neurons express ASIC3, including unmyelinated (neurofilament poor) and small diameter myelinated (neurofilament rich) neurons that are likely to be C and Aδ nerve fibers respectively. Electrophysiological recordings revealed that application of APET×2 to the marrow cavity inhibited carrageenan-induced spontaneous activity of C and Aδ fiber bone afferent neurons. APET×2 also inhibited carrageenan-induced sensitization of Aδ and C fiber bone afferent neurons to mechanical stimulation, but had no effect on the sensitivity of bone afferent neurons in the absence of inflammation. This evidence supports a role for ASIC3 in the pathogenesis of pain associated with inflammation of the bone.

The neurohypophysial hormone oxytocin (OXT) is synthesized in the hypothalamic paraventricular and supraoptic nuclei. Recently, some studies have considered OXT to be important in sensory modulation and that the OXT protein is upregulated by acute and chronic nociception. However, the mechanism by which OXT is upregulated in neurons is unknown. In this study, we examined the resting membrane potentials and excitatory postsynaptic currents in OXT-ergic neurons in the paraventricular nucleus in adjuvant arthritis rat model, a model of chronic inflammation, using whole-cell patch-clamping. Transgenic rats expressing OXT and monomeric red fluorescent protein 1 (mRFP1) fusion protein to visualize the OXT-ergic neurons were used, and the OXT-mRFP1 transgenic rat model of adjuvant arthritis was developed by injection of heat-killed . Furthermore, the feedback system of synthesized OXT was also examined using the OXT receptor antagonist L-368,899. We found that the resting membrane potentials and frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-monomeric red fluorescent protein 1 neurons in the paraventricular nucleus were significantly increased in adjuvant arthritis rats. Furthermore, L-368,899 dose-dependently increased the frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-ergic neurons. Following bath application of the GABA receptor antagonist picrotoxin and the cannabinoid receptor 1 antagonist AM 251, L-368,899 still increased the frequency of miniature excitatory postsynaptic currents. However, following bath application of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride, L-368,899 did not alter the miniature excitatory postsynaptic current frequency. Thus, it is suggested that OXT-ergic neuron activity is upregulated via an increase in glutamate release, and that the upregulated OXT neurons have a feedback system with released endogenous OXT. It is possible that nitric oxide, but not GABA, may contribute to the feedback system of OXT neurons in chronic inflammation.

Loss of the sense of touch or numbness in fingertips and toes is one of the earliest sensory dysfunctions in patients receiving chemotherapy with anti-cancer drugs such as vincristine. However, mechanisms underlying this chemotherapy-induced sensory dysfunction is poorly understood. Whisker hair follicles are tactile organs in non-primate mammals which are functionally equivalent to human fingertips. Here we used mouse whisker hair follicles as a model system to explore how vincristine treatment induces the loss of the sense of touch. We show that chronic treatment of mice with vincristine impaired in vivo whisker tactile behavioral responses. In vitro electrophysiological recordings made from whisker hair follicle afferent nerves showed that mechanically evoked whisker afferent impulses were significantly reduced following vincristine treatment. Furthermore, patch-clamp recordings from Merkel cells of whisker hair follicles revealed a significant reduction of mechanically activated currents via Piezo2 channels in Merkel cells. Collectively, our results suggest that Piezo2 channel dysfunction in Merkel cells contribute to the loss of the sense of touch following the chemotherapy treatment regimen with vincristine.

Recent studies indicate that presynaptic long-term potentiation (pre-LTP) in the anterior cingulate cortex (ACC) may contribute to chronic pain-related anxiety. In addition to the ACC, the insular cortex (IC) has also been indicated in chronic pain and its related emotional disorders. In the present study, we used a 64-channel multielectrode dish (MED64) system to record pre-LTP in the IC. We showed that low-frequency stimulation paired with a GluK1-containing kainate receptor agonist induced NMDA receptor-independent pre-LTP in the IC of wide-type (WT) mice. This form of pre-LTP was blocked in the IC of adenylyl cyclase subtype 1 (AC1) KO mice. Furthermore, a selective AC1 inhibitor NB001 blocked pre-LTP in the IC with a dose-dependent manner. Taken together, our results suggest that AC1 contributes to pre-LTP in the IC of adult mice and NB001 may produce anxiolytic effects by inhibiting pre-LTP in the ACC and IC.

A well-recognized relationship exists between ageing and increased susceptibility to chronic pain conditions, underpinning the view that pain signaling pathways differ in aged individuals. Yet despite the higher prevalence of altered pain states among the elderly, the majority of preclinical work studying mechanisms of aberrant sensory processing are conducted in juvenile or young adult animals. This mismatch is especially true for electrophysiological studies where patch clamp recordings from aged tissue are generally viewed as particularly challenging. In this study we have undertaken an electrophysiological characterization of spinal dorsal horn neurons in young adult (3-4 months) and aged (28-32 months) mice. We show that patch clamp data can be routinely acquired in spinal cord slices prepared from aged animals and that the excitability properties of aged dorsal horn neurons differ from recordings in tissue prepared from young animals. Specifically, aged dorsal horn neurons more readily exhibit repetitive action potential discharge, indicative of a more excitable phenotype. This observation was accompanied by a decrease in the amplitude and charge of spontaneous excitatory synaptic input to dorsal horn neurons and an increase in the contribution of GABAergic signalling to spontaneous inhibitory synaptic input in aged recordings. While the functional significance of these altered circuit properties remains to be determined, future work should seek to assess if such features may render the aged dorsal horn more susceptible to aberrant injury or disease induced signaling and contribute to increased pain in the elderly.

The formation of neuromas involves expansion of the cellular components of peripheral nerves. The onset of these disorganized tumors involves activation of sensory nerves and neuroinflammation. Particularly problematic in neuroma is arborization of axons leading to extreme, neuropathic pain. The most common sites for neuroma are the ends of transected nerves following injury; however, this rodent model does not reliably result in neuroma formation. In this study, we established a rodent model of neuroma in which the sciatic nerve was loosely ligated with two chromic gut sutures [1]. This model formed neuromas reliably (~95%), presumably through activation of the neural inflammatory cascade. Resulting neuromas had a disorganized structure and a significant number of replicating cells. Quantification of changes in perineurial and Schwann cells showed a significant increase in these populations. Immunohistochemical analysis showed the presence of β-tubulin 3 (TUJ1) in the rapidly expanding nerve and a decrease in neurofilament heavy chain compared to the normal nerve, suggesting the axons forming a disorganized structure. Measurement of the permeability of the blood-nerve barrier (BNB) shows that it opened almost immediately and remained open as long as 10 days. Studies using an antagonist of the 3-adrenergic receptor (ADRβ3) (L-748,337) or cromolyn showed a significant reduction in tumor size and cell expansion as determined by flow cytometry, with an improvement in the animal's gait detected using a Catwalk system. Previous studies in our laboratory have shown that heterotopic ossification (HO) is also a result of the activation of neuroinflammation. Since HO and neuroma often occur together in amputees, they were induced in the same limbs of the study animals. More heterotopic bone was formed in animals with neuromas as compared to those without. These data collectively suggest that perturbation of early neuroinflammation with compounds such as L-748,337 and cromolyn may reduce formation of neuromas.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease with many known structural and functional changes in the central nervous system (CNS). A well-recognized, but poorly understood, complication of MS is chronic pain. Little is known regarding the influence of sex on the development and maintenance of MS-related pain. This is important to consider, as MS is a predominantly female disease. Using the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we demonstrate sex differences in measures of spinal cord inflammation and plasticity that accompany tactile hypersensitivity. While we observed substantial inflammatory activity in both sexes, only male EAE mice exhibit robust staining of axonal injury markers and increased dendritic arborisation in morphology of deep dorsal horn neurons. We propose that tactile hypersensitivity in female EAE mice may be more immune-driven, while pain in male mice with EAE may rely more heavily on neurodegenerative and plasticity-related mechanisms. Morphological and inflammatory differences in the spinal cord associated with pain early in EAE progression supports the idea of differentially regulated pain pathways between the sexes. Results from this study may indicate future sex-specific targets that are worth investigating for their functional role in pain circuitry.

The anterior cingulate cortex (ACC) modulates emotional responses to pain. Whereas, the caudal ACC (cACC) promotes expression of pain affect, the rostral ACC (rACC) contributes to its suppression. Both subdivisions receive glutamatergic innervation, and the present study evaluated the contribution of NMDA receptors within these subdivisions to rats' expression of pain affect. Vocalizations that follow a brief noxious tail shock (vocalization afterdischarges, VAD) are a validated rodent model of pain affect. The threshold current for eliciting VAD was increased in a dose-dependent manner by injecting NMDA into the rACC, but performance (latency, amplitude and duration) at threshold was not altered. Alternately, the threshold current for eliciting VAD was not altered following injection of NMDA into the cACC, but its amplitude and duration at threshold were increased in a dose-dependent manner. These effects were limited to Cg1 of the rACC and cACC, and blocked by pre-treatment of the ACC with the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP-5). These findings demonstrate that NMDA receptor agonism within the cACC and rACC either increase or decrease emotional responses to noxious stimulation, respectively. Perspective: NMDA receptor activation of the rostral and caudal anterior cingulate cortex respectively inhibited or enhanced rats' emotional response to pain. These findings mirror those obtained from human neuroimaging studies; thereby, supporting the use of this model system in evaluating the contribution of ACC to pain affect.