Animal Studies

The progressive increase in the prevalence of obesity in the population has resulted in increased healthcare costs and demands. Recent studies have revealed a positive correlation between pain and obesity, although the underlying mechanisms still remain unknown. Here, we aimed to clarify the role of microglia in altered pain behaviors induced by a high-fat diet (HFD) in male mice. We found that C57BL/6CR mice on a HFD exhibited spinal microglial reaction (increased cell number and up-regulated expression of p-p38 and CD16/32), increased tumor necrosis factor-α (TNF-α) mRNA and brain-derived neurotrophic factor (BDNF) protein as well as a polarization of spinal microglial toward a pro-inflammatory phenotype. Moreover, we found that when applied PLX3397 (a selective colony-stimulating factor-1 receptor (CSF1R) kinase inhibitor) to eliminate microglia of HFD-induced obesity mice, inflammation in the spinal cord was rescued, as were abnormal pain hypersensitivity. Intrathecal injection of Mac-1-saporin (a saporin-conjugated anti-mac1 antibody) resulted in decreased microglia and attenuated both mechanical allodynia and thermal hyperalgesia in HFD-fed mice. These results indicate that the pro-inflammatory functions of spinal microglia have a special relevance to abnormal pain hypersensitivity in HFD-induced obesity mice. In conclusion, our data suggest that HFD induces a classical reaction of microglia, characterized by an enhanced phosphorylation of p-38 and increased CD16/32 expression, which may in part contribute to increased nociceptive responses in HFD-induced obesity mice.

NGF is increased in intervertebral discs (IVDs) after disc injury and anti-NGF therapy improves low back pain in humans. Further, M1 and M2 macrophage subtypes play a role in degenerative IVD injury. We examined M1 and M2 macrophage markers and NGF and cytokine expression in IVD-derived cells from control and IVD-injured mice for 28 days following injury. Ngf mRNA expression was increased 1 day after injury in injured compared to control mice, and persisted for up to 28 days. Flow cytometric analysis demonstrated that the proportion of F4/80 + CD11b + cells was significantly increased from 1 day after injury for up to 28 days in injured compared to control mice. mRNA expression of M1 macrophage markers Tnfa, Il1b, and Nos2 was significantly increased 1 day after injury in injured compared to control mice, before gradually decreasing. At 28 days, no significant difference was observed in M1 markers. The M2a marker, Ym1, was significantly increased 1 day after injury in injured compared to control mice, while M2a and M2c markers Tgfb and Cd206 were significantly increased 7, 14, and 28 days after injury. TNF-α and TGF-β stimulated Ngf mRNA and NGF protein expression in IVD cells. Our results suggest that TNF-α and TGF-β may stimulate NGF production under inflammatory and non-inflammatory conditions following IVD injury. As TNF-α and TGF-β are produced by M1 and M2 macrophages, further investigations are needed to reveal the role of macrophages in NGF expression following IVD injury. Our results may aid in developing treatments for IVD-related LBP pathology. This article is protected by copyright. All rights reserved.

The present study aimed at investigating both the early and long-term effects of maternal deprivation as well as gender on neuromotor reflexes, anxiety behavior and thermal nociceptive responses. A total of 64 Wistar rats pups (32 males, 32 females) were utilized and were deprived of their mother for 3 h/daily, from postnatal day 1 (P1) until P10. Successively, animals were divided into 2 groups: control group (C) – pups no subjected to intervention; and the maternal-deprived group (MD): pups subjected to maternal deprivation. The neuromotor reflexes were evaluated through the righting reflex and negative geotaxis tests; the exploratory behavior by open field test (OFT); the anxiety-like behavior by elevated plus-maze test (EPM); the thermal nociceptive responses byhot plate (HP) and tail-flick (TFL) tests. All the animals subjected to maternal deprivation showed a delayed reflex response at P8 in the negative geotaxis test. In contrast, the OFT at P20 identified an effect of gender on the outer crossings and grooming as well as an interaction between gender and maternal deprivation on latency. Additionally, effect of maternal deprivation in the open and closed arms as well as gender effect in the protected head-dipping (PHD) and non-protected head-dipping (NPHD) were observed at P20 (EPM). In contrast, there were a gender effect on latency and an interaction between gender and maternal deprivation on rearing at P42. Moreover, in nociceptive tests was observed an analgesic effect induced by maternal deprivation; however, in the TFL test, only deprived females showed this effect. Surprisingly, only control animals presented an ontogeny nociceptive effect in the HP testat P21 and P43, which may be related to an increase in the inhibitory nociceptive pathways throughout life. In this way, we suggest maternal deprivation to be able to anticipate the maturation of the inhibitory nociceptive pathway. In conclusion, maternal deprivation induced a delayed reflex response at P8 and altered the anxiety and nociceptive behaviors according to the time after exposure to this stressor, in a gender-specific manner.

Inhibitory glycinergic transmission in adult spinal cord is primarily mediated by glycine receptors (GlyRs) containing the α1 subunit. Here, we found that α1ins, a longer α1 variant with 8 amino acids inserted into the intracellular large loop (IL) between transmembrane (TM)3 and TM4 domains, was expressed in the dorsal horn of the spinal cord, distributed at inhibitory synapses, and engaged in negative control over nociceptive signal transduction. Activation of metabotropic glutamate receptor 5 (mGluR5) specifically suppressed α1ins-mediated glycinergic transmission and evoked pain sensitization. Extracellular signal-regulated kinase (ERK) was critical for mGluR5 to inhibit α1ins. By binding to a D-docking site created by the 8-amino-acid insert within the TM3-TM4 loop of α1ins, the active ERK catalyzed α1ins phosphorylation at Ser380, which favored α1ins ubiquitination at Lys379 and led to α1ins endocytosis. Disruption of ERK interaction with α1ins blocked Ser380 phosphorylation, potentiated glycinergic synaptic currents, and alleviated inflammatory and neuropathic pain. These data thus unraveled a novel, to our knowledge, mechanism for the activity-dependent regulation of glycinergic neurotransmission.

The cold and menthol sensitive Transient Receptor Potential Melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein coupled receptors (GPCRs) by pro-inflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of G to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P or PIP] for activity. It was claimed however that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here we show that supplementing the whole cell patch pipette with PI(4,5)P reduced inhibition of TRPM8 by activation of G-coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated Phospholipase C (PLC) and decreased plasma membrane PI(4,5)P levels in these neurons. PI(4,5)P also reduced inhibition of TRPM8 by activation of heterologously expressed G-coupled muscarinic M1 receptors. Co-expression of a constitutively active G protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein decreasing PI(4,5)P levels using a voltage sensitive 5'-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that upon GPCR activation, G binding reduces the apparent affinity of TRPM8 for PI(4,5)P and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P levels.Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold- and menthol sensitive TRPM8 ion channels. Most inflammatory mediators act via GPCR-s that activate the Phospholipase C pathway leading to the hydrolysis of PI(4,5)P How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here we propose that direct binding of G both reduces TRPM8 activity, and sensitizes the channel to inhibition by decreased levels of its cofactor PI(4,5)P Our data demonstrate the convergence of two downstream effectors of receptor activation G and PI(4,5)P hydrolysis in regulation of TRPM8.

Mechanical allodynia is a cardinal feature of pathological pain. Recent work has demonstrated the necessity of A[graphic1] low-threshold mechanoreceptors (Aβ-LTMRs) for mechanical allodynia-like behaviors in mice, but it remains unclear whether these neurons are sufficient to produce pain under pathological conditions. Thus, we generated a transgenic mouse in which channelrhodopsin-2 (ChR2) is conditionally expressed in Vesicular Glutamate Transporter 1 (Vglut1) sensory neurons (Vglut1-ChR2), which is a heterogeneous population of large-sized sensory neurons with features consistent with (A[graphic3]-LTMRs). In naive male Vglut1-ChR2 mice, transdermal hindpaw photostimulation evoked withdrawal behaviors in an intensity- and frequency-dependent manner, which were abolished by local anesthetic and also selective A-fiber blockade. Surprisingly, male Vglut1-ChR2 mice did not show significant differences in light-evoked behaviors or real-time aversion after nerve injury, despite marked hypersensitivity to punctate mechanical stimuli. Thus, we conclude that optogenetic activation of cutaneous Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice. Mechanical allodynia, wherein innocuous touch is perceived as pain, is a common feature of pathological pain. To test the contribution of low-threshold mechanoreceptors to nerve injury-induced mechanical allodynia, we generated and characterized a new transgenic mouse (Vglut1-ChR2) to optogenetically activate cutaneous Vglut1-positive LTMRs. Using this mouse, we found that light-evoked behaviors were unchanged by nerve injury, which suggests that activation of Vglut1-positive LTMRs alone is not sufficient to produce pain. The Vglut1-ChR2 mouse will be broadly useful for the study of touch, pain and itch.

Dysfunction of the prefrontal-hippocampal circuit has been identified as a leading cause to pain-related working-memory (WM) deficits. However, the underlying mechanisms remain poorly determined. To address this issue, we implanted multichannel arrays of electrodes in the prelimbic cortex (PL-mPFC), and in the dorsal hippocampal CA1 field (dCA1) to record the neural activity during the performance of a delayed non-match to sample (DNMS) task. The prefrontal-hippocampal connectivity was selectively modulated by bidirectional optogenetic inhibition or stimulation of local PL-mPFC glutamatergic calcium/calmodulin-dependent protein kinase-II alpha (CaMKIIα) expressing neurons during the DNMS task delay-period. The within-subject behavioral performance was assessed using a persistent neuropathic pain model – spared nerve injury (SNI). Our results showed that the induction of the neuropathic pain condition affects the interplay between PL-mPFC and dCA1 regions in a frequency-dependent manner, and that occurs particularly across theta oscillations while rats performed the task. In SNI-treated rats, this disruption was reversed by the selective optogenetic inhibition of PL-mPFC CaMKIIα-expressing neurons during the last portion of the delay-period, but without any significant effect on pain responses. Finally, we found that prefrontal-hippocampal theta connectivity is strictly associated with higher performance levels. Together, our findings suggest that PL-mPFC CaMKIIα-expressing neurons could be modulated by painful conditions and their activity may be critical for prefrontal-hippocampal connectivity during WM processing.