Animal Studies

Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that is inhibited by Gβγ subunits liberated following activation of Gα protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gα and Gα Activation of the G-coupled adenosine 2B receptor and the G-coupled muscarinic acetylcholine M1 receptor inhibited the activity of heterologously expressed TRPM3 in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C-terminus of β-adrenergic receptor kinase-1) was co-expressed with TRPM3. In neurons isolated from mouse dorsal root ganglia (DRG), native TRPM3 channels were inhibited by activating G-coupled prostaglandin-EP2 and G-coupled bradykinin B2 (BK2) receptors. The G inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gα, or through the major protein kinases activated downstream of GPCR activation. When DRG neurons were dialysed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat-hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat-hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in wildtype mice and did not develop in mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and , and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gα-coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gα, we examined whether activation of G- and G-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of G- and G-coupled GPCRs in recombinant cells and native sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- and G-coupled receptors inhibit TRPM3 , thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

Neonatal tissue injury disrupts the balance between primary afferent-evoked excitation and inhibition onto adult spinal projection neurons. However, whether this reflects cell type-specific alterations at synapses onto ascending projection neurons, or rather is indicative of global changes in synaptic signaling across the mature superficial dorsal horn (SDH), remains unknown. Therefore the present study investigated the effects of neonatal surgical injury on primary afferent synaptic input to adult mouse SDH interneurons using in vitro patch clamp techniques. Hindpaw incision at postnatal day (P) 3 significantly diminished total primary afferent-evoked glutamatergic drive to adult Gad67-GFP and non-GFP neurons, and reduced their firing in response to sensory input, in both males and females. Early tissue damage also shaped the relative prevalence of monosynaptic A- vs. C-fiber mediated input to mature GABAergic neurons, with an increased prevalence of Aβ- and Aδ-fiber input observed in neonatally-incised mice compared to naïve littermate controls. Paired presynaptic and postsynaptic stimulation at an interval that exclusively produced spike timing-dependent long-term potentiation (t-LTP) in projection neurons predominantly evoked NMDAR-dependent long-term depression (t-LTD) in naïve Gad67-GFP interneurons. Meanwhile, P3 tissue damage enhanced the likelihood of t-LTP generation at sensory synapses onto the mature GABAergic population, and increased the contribution of Ca-permeable AMPARs to the overall glutamatergic response. Collectively, the results indicate that neonatal injury suppresses sensory drive to multiple subpopulations of interneurons in the adult SDH, which likely represents one mechanism contributing to reduced feedforward inhibition of ascending projection neurons, and the priming of developing pain pathways, following early life trauma.Mounting clinical and preclinical evidence suggests that neonatal tissue damage can result in long-term changes in nociceptive processing within the CNS. While recent work has demonstrated that early life injury weakens the ability of sensory afferents to evoke feedforward inhibition of adult spinal projection neurons, the underlying circuit mechanisms remain poorly understood. Here we demonstrate that neonatal surgical injury leads to persistent deficits in primary afferent drive to both GABAergic and presumed glutamatergic neurons in the mature superficial dorsal horn (SDH), and modifies activity-dependent plasticity at sensory synapses onto the GABAergic population. The functional denervation of spinal interneurons within the mature SDH may contribute to the 'priming' of developing pain pathways following early life injury.

Neuropathy is a common, morbid complication of the metabolic syndrome, prediabetes, and diabetes. Recent studies have indicated a potential role for the immune system in the development of neuropathy. In particular, toll-like receptors (TLR) 2 and 4 have been linked to metabolic dysfunction, and blocking TLR4 is proposed as a treatment for neuropathic pain. In the current study, we investigated the role of the immune system, particularly TLRs 2 and 4, in the pathogenesis and progression of neuropathy. Sural or sciatic nerve gene expression arrays from humans and murine neuropathy models of prediabetes and diabetes were first analyzed to identify differentially expressed TLR2- and TLR4-associated genes within the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. We observed that genes associated with TLRs 2 and 4, particularly lipopolysaccharide binding protein (LPB) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), were dysregulated across species and across multiple murine models of prediabetic and diabetic neuropathy. To further understand the role of these pathways in vivo, TLR 2 and 4 global knockout mice placed on a 60% high fat diet (HFD-TLR2/4) were compared with wild type (WT) mice on a high fat diet (HFD-WT) and WT controls on a standard diet (CON). Mice then underwent metabolic, neuropathic, and immunological phenotyping at two time points to assess the impact of TLR signaling on neuropathy and immunity during metabolic dysfunction over time. We found that HFD-TLR2/4 and HFD-WT mice weighed more than CON mice but did not have increased fasting blood glucose levels. Despite normal blood glucose levels, HFD-TLR2/4 mice eventually developed neuropathy at the later time point (28 wks of age) but were somewhat protected from neuropathy at the early time point (16 wks of age) as measured by shorter hind paw withdraw latencies. This is in contrast to HFD-WT mice which developed neuropathy within 11 wks of being placed on a high fat diet and were neuropathic by all measures at both the early and late time points. Finally, we immunophenotyped all three mouse groups at the later time point and found differences in the number of peripheral blood Ly6C-myeloid cells as well as F4/80+ expression. These results indicate that TLR signaling influences early development of neuropathy in sensory neurons, potentially via immune modulation and recruitment.

Current strategies to enhance regeneration of peripheral neurons involve broad activation of sensory, autonomic, and motor axons. Peripheral neuron regeneration is limited in persons with damage or disease of peripheral axons. Here, we provide evidence that subtoxic activation of TRPV1 channels in sensory neurons is associated with activation of growth and subtle changes in skin reinnervation. We identify a bidirectional, dose-related impact of capsaicin, a TRPV1 agonist, on sensory neurons and their axons with rises in their outgrowth plasticity at low doses and toxic neurodegeneration at high doses. Moreover, its impact on growth added to that of preconditioning. Neither outcome was observed in TRPV1 null neurons. We confirmed that low dose activation was associated with rises in neuronal calcium, as well as rises in TRPV1 mRNA transcripts. In mice with a sciatic nerve crush followed by a single application of capsaicin directly to the injury site, there was no impact on motor or myelinated axon recovery but there was evidence of better recovery of thermal sensation toward baseline with hyperalgesia. Moreover, skin reinnervation by epidermal axons approached contralateral levels. TRPV1 null mice displayed loss of thermal sensation during later recovery. In sensory axons innervating the pinna of the ear, local capsaicin rendered early axon loss followed by later hyperinnervation. Taken together, TRPV1 activation alters the regenerative behavior of adult neurons and their axons both in vitro and during epidermal reinnervation in vivo. The findings identify a selective manipulation that augments cutaneous innervation by thermosensitive axons.

Human and animal imaging studies demonstrated that chronic pain profoundly alters the structure and the functionality of several brain regions. Herein, we conducted a longitudinal and multimodal study to assess how chronic pain affects the brain. Using the Spared Nerve Injury model (SNI) which promotes both long-lasting mechanical and thermal allodynia/hyperalgesia but also pain-associated comorbidities, we showed that neuropathic pain deeply modified the intrinsic organization of the brain functional network one and two months after injury. We found that both functional metrics and connectivity of the part A of the retrosplenial granular cortex (RSgA) were significantly correlated with the development of neuropathic pain behaviours. Additionally, we found that the functional RSgA connectivity to the subiculum and the prelimbic system are significantly increased in SNI animals and correlated with peripheral pain thresholds. These brain regions were previously linked to the development of comorbidities associated with neuropathic pain. Using a Voxel-Based Morphometry approach, we showed that neuropathic pain induced a significant increase of the grey matter concentration within the RSgA, associated with a significant activation of both astrocytes and microglial cells. Together, functional and morphological imaging metrics of the RSgA could be used as a predictive biomarker of neuropathic pain.

Neuropathic pain represents one of the most common complications associated with diabetes mellitus (DM) that impacts quality of life. Accumulating studies have highlighted the involvement of microRNAs (miRNAs) in DM. Thus, the current study aimed to investigate the roles of microRNA-155 (miR-155) in diabetic peripheral neuropathy (DPN). In vitro DPN models were established using rat Schwann cells (SCs) by treatment with 5.5 mM glucose. Gain- or loss-of-function studies were conducted to determine the effect of miR-155 on Nrf2, cellular function, reactive oxygen species, and inflammation. Rat DNP models were established by streptozotocin injection and damage of sciatic nerve. Next, miR-155 antagomir or agomir was employed to investigate the effects associated with miR-155 on motor and sciatic nerve conduction velocity (MNCV, SNCV), angiogenesis and inflammatory response in vivo. Nrf2 was identified to be a target of miR-155 by dual-luciferase reporter gene assay. Silencing of miR-155 or restoration of Nrf2 promoted cell proliferation, inhibited apoptosis and alleviated inflammation in vitro. miR-155 antagomir-induced inhibition increased MNCV and SNCV, strengthened angiogenesis and alleviated inflammation in DPN rats. Additionally, the effects exerted by miR-155 were reversed when Nrf2 was restored both in vitro and in vivo. Taken together, the key findings of our study provide evidence indicating that miR-155 targeted and suppressed Nrf2 in DPN. miR-155 silencing was found to alleviate sciatic nerve injury in DPN, highlighting its potential as a therapeutic target for DPN.