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3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is a selective μ-opioid receptor agonist originally synthesized as a prospective analgesic drug. Several times more potent than morphine, U-47700 has high abuse potential and may cause clinical neurotoxicity, euphoria, respiratory depression and occasional mortality. U-47700 also evokes analgesia, sedation and euphoria-like states in both humans and rodents. Despite the growing use and abuse of U-47700, its psychopharmacological and toxicological profiles remain poorly understood. The zebrafish (Danio rerio) is rapidly becoming a popular aquatic model organism for CNS disease modeling and drug discovery. Here, we examine acute (1, 5, 10, 25 and 50 mg/L for 20-min) and chronic (0.1, 0.5 and 1 mg/L for 14 days) effects of U-47700 in adult zebrafish. Overall, we found overt sedation by acute, and hyperlocomotion with an anxiolytic-like action by chronic, drug treatments. Acute treatment with 1 and 10 mg/L U-47700 also resulted in detectable amounts of this drug in the brain samples. Collectively, these findings emphasize a complex dose- and treatment-dependent CNS profile of U-47700 following its acute and chronic administration. Our study also supports high sensitivity of zebrafish to U-47700, and suggests these aquatic models as promising in-vivo screens for probing CNS effects evoked by novel synthetic opioid drugs.
Learn More >Mice cohabiting with a conspecific in chronic pain display anxiogenesis in the elevated plus-maze (EPM). Given that the anterior cingulate (ACC) and insular (InC) cortices play a role in the modulation of anxiety, pain, and emotional contagion, we investigated (a) the FosB activation in both brain areas and (b) the effects of intra-ACC or -InC injection of cobalt chloride (CoCl, a synaptic blocker), on the anxiety of mice cohabiting with a cagemate suffering pain. Twenty-one days after birth, male Swiss mice were housed in pairs for 14 days to establish familiarity. On the 14th day, mice were divided into two groups: cagemate sciatic nerve constriction (CNC; i.e., one animal of each pair was subjected to sciatic nerve constriction), and cagemate sham (CS; i.e., a similar procedure but without suffering nerve constriction). After that, both groups were housed again with the same pairs for the other 14 days. On the 28th day, mice had their brains removed for the immunoassays analyses (Exp. 1). For experiments 2 and 3, on the 23rd day, the cagemates received guide cannula implantation bilaterally in the ACC or InC and, on the 28th day, they received local injections of saline or CoCl, and then were exposed to the EPM. Results showed that cohabitation with a conspecific with chronic pain decreases and increases neuronal activation (FosB) within the ACC and InC, respectively. Intra-ACC or InC injection of CoCl reversed the anxiogenic effect in those animals that cohabited with a conspecific in chronic pain. ACC and InC seem to modulate anxiety induced by emotional contagion in animals cohabitating with a conspecific suffering pain.
Learn More >The lateral parabrachial nucleus (LPBN) is known to relay noxious information to the amygdala for processing affective responses. However, it is unclear whether the LPBN actively processes neuropathic pain characterized by persistent hyperalgesia with aversive emotional responses. Here we report that neuropathic pain-like hypersensitivity induced by common peroneal nerve (CPN) ligation increases nociceptive stimulation-induced responses in glutamatergic LPBN neurons. Optogenetic activation of GABAergic LPBN neurons does not affect basal nociception, but alleviates neuropathic pain-like behavior. Optogenetic activation of glutamatergic or inhibition of GABAergic LPBN neurons induces neuropathic pain-like behavior in naïve mice. Inhibition of glutamatergic LPBN neurons alleviates both basal nociception and neuropathic pain-like hypersensitivity. Repetitive pharmacogenetic activation of glutamatergic or GABAergic LPBN neurons respectively mimics or prevents the development of CPN ligation-induced neuropathic pain-like hypersensitivity. These findings indicate that a delicate balance between excitatory and inhibitory LPBN neuronal activity governs the development and maintenance of neuropathic pain.
Learn More >A large body of animal and human studies indicate that blocking peripheral calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling pathways may prevent migraine episodes and reduce headache frequency. To investigate whether recurring migraine episodes alter the strength of CGRP and PACAP signaling in trigeminal ganglion (TG) neurons, we compared the number of TG neurons that respond to CGRP and to PACAP (CGRP-R and PACAP-R, respectively) under normal and chronic migraine-like conditions. In a mouse model of chronic migraine, repeated nitroglycerin (NTG) administration significantly increased the number of CGRP-R and PACAP-R neurons in TG but not dorsal root ganglia. In TG neurons that express endogenous αCGRP, repeated NTG led to a 7-fold increase in the number of neurons that respond to both CGRP and PACAP (CGRP-R&PACAP-R). The majority of these neurons were unmyelinated C-fiber nociceptors. This suggests that a larger fraction of CGRP signaling in TG nociceptors may be mediated through the autocrine mechanism, and the release of endogenous αCGRP can be enhanced by both CGRP and PACAP signaling pathways under chronic migraine condition. The number of CGRP-R&PACAP-R TG neurons was also increased in a mouse model of post-traumatic headache (PTH). Interestingly, low-dose interleukin-2 treatment, which completely reverses chronic migraine- and PTH-related behaviors in mouse models, also blocked the increase in both CGRP-R and PACAP-R TG neurons. Together, these results suggest that inhibition of both CGRP and PACAP signaling in TG neurons may be more effective in treating chronic migraine and PTH than targeting individual signaling pathways.
Learn More >A better understanding of neural pain processing and of the development of pain over time, is critical to identify objective measures of pain and to evaluate the effect of pain alleviation therapies. One issue is, that the brain areas known to be related to pain processing are not exclusively responding to painful stimuli, and the neuronal activity is also influenced by other brain areas. Functional connectivity reflects synchrony or covariation of activation between groups of neurons. Previous studies found changes in connectivity days or weeks after pain induction. However, less in known on the temporal development of pain. Our objective was therefore to investigate the interaction between the anterior cingulate cortex (ACC) and primary somatosensory cortex (SI) in the hyperacute (minute) and sustained (hours) response in an animal model of neuropathic pain. Intra-cortical local field potentials (LFP) were recorded in 18 rats. In 10 rats the spared nerve injury model was used as an intervention. The intra-cortical activity was recorded before, immediately after, and three hours after the intervention. The interaction was quantified as the calculated correlation and coherence. The results from the intervention group showed a decrease in correlation between ACC and SI activity, which was most pronounced in the hyperacute phase but a longer time frame may be required for plastic changes to occur. This indicated that both SI and ACC are involved in hyperacute pain processing.
Learn More >Neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2) is a member of the E3 ubiquitin ligase family that is highly expressed in sensory neurons and involved in pain modulation via downregulation of ion channels in excitable membranes. Ubiquitination involving Nedd4-2 is regulated by adenosine monophosphate-activated protein kinase (AMPK), which is impaired in the dorsal root ganglion (DRG) neurons of db/db mice. AMPK negatively regulates the expression of transient receptor potential ankyrin 1 (TRPA1), a recognised pain sensor expressed on the membrane of DRG neurons, consequently relieving mechanical allodynia in db/db mice. Herein, we studied the involvement of Nedd4-2 in painful diabetic neuropathy and observed that Nedd4-2 negatively regulated diabetic mechanical allodynia. Nedd4-2 was co-expressed with TRPA1 in mouse DRG neurons. Nedd4-2 was involved in TRPA1 ubiquitination, this ubiquitination, as well as Nedd4-2-TRPA1 interaction, was decreased in db/db mice. Moreover, Nedd4-2 levels were decreased in db/db mice, while an abnormal intracellular distribution was observed in short-term high glucose-cultured DRG neurons. AMPK activators not only restored Nedd4-2 distribution but also increased Nedd4-2 expression. These findings demonstrate that Nedd4-2 is a potent regulator of TRPA1, and that the abnormal expression of Nedd4-2 in DRG neurons contributes to diabetic neuropathic pain.
Learn More >Chronic postsurgical pain (CPSP) is a serious issue for many postoperative patients. Though there are numerous treatment options for the prevention of CPSP, none of them is optimal as the mechanisms of the transition from acute to chronic postoperative pain have not been elucidated. Ketamine and opioids have been administered for chronic postoperative pain treatment but induce severe adverse reactions and/or physical dependency. Here, we examined whether pre-administration of the nonselective N-methyl-d-aspartate (NMDA) receptor antagonist magnesium sulfate attenuates CPSP behavior and alters the expression of glutamate ionotropic receptor NMDA type subunit 1a (Grin1 mRNA) in a rat skin/muscle incision and retraction (SMIR) model. We assessed the effects of a single subcutaneous magnesium sulfate injection on nociceptive behaviors including guarding pain, mechanical hyperalgesia, and heat hypersensitivity in rats after SMIR surgery. We used reverse transcription-quantitative PCR (RT-qPCR) to evaluate Grin1 mRNA expression in the dorsal horn of the spinal cord on postoperative day 14. Compared with the vehicle, magnesium sulfate administration before SMIR surgery reduced mechanical hyperalgesia for 17 d Grin1 gene expression was significantly higher on the ipsilateral side than the contralateral side (P = 0.001) on postoperative day 14. The magnesium sulfate injection prevented Grin1 mRNA upregulation in the spinal cord dorsal horn. A single magnesium sulfate injection mitigated SMIR-induced mechanical hyperalgesia possibly by modulating Grin1 expression. Preoperative magnesium sulfate administration could prove to be a simple and safe CPSP treatment.
Learn More >This study aimed to examine the temporal activation of NF-κB and its relationship to the development of pain-related sensitivity and behavioral changes in a non-invasive murine knee loading model of PTOA.
Learn More >Postoperative cognitive dysfunction (POCD) is the collection of cognitive impairments, lasting days to months, experienced by individuals following surgery. Persistent POCD is most commonly experienced by older individuals and is associated with a greater vulnerability to developing Alzheimer's disease, but the underlying mechanisms are not known. It is known that laparotomy (exploratory abdominal surgery) in aged rats produces memory impairments for 4 days. Here we report that postsurgical treatment with morphine extends this deficit to at least 2 months while having no effects in the absence of surgery. Indeed, hippocampal-dependent long-term memory was impaired 2, 4, and 8 weeks postsurgery only in aged, morphine-treated rats. Short-term memory remained intact. Morphine is known to have analgesic effects via μ-opioid receptor activation and neuroinflammatory effects through Toll-like receptor 4 activation. Here we demonstrate that persistent memory deficits were mediated independently of the μ-opioid receptor, suggesting that they were evoked through a neuroinflammatory mechanism and unrelated to pain modulation. In support of this, aged, laparotomized, and morphine-treated rats exhibited increased gene expression of various proinflammatory markers (IL-1β, IL-6, TNFα, NLRP3, HMGB1, TLR2, and TLR4) in the hippocampus at the 2-week time point. Furthermore, central blockade of IL-1β signaling with the specific IL-1 receptor antagonist (IL-1RA), at the time of surgery, completely prevented the memory impairment. Finally, synaptophysin and PSD95 gene expression were significantly dysregulated in the hippocampus of aged, laparotomized, morphine-treated rats, suggesting that impaired synaptic structure and/or function may play a key role in this persistent deficit. This instance of long-term memory impairment following surgery closely mirrors the timeline of persistent POCD in humans and may be useful for future treatment discoveries.
Learn More >Cancer-induced bone pain (CIBP) is a complex condition, comprising components of inflammatory and neuropathic processes, but changes in the physiological response profiles of bone-innervating and cutaneous afferents remain poorly understood. We used a combination of retrograde labelling and in vivo calcium imaging of bone marrow-innervating dorsal root ganglia (DRG) neurons to determine the contribution of these cells in the maintenance of CIBP. We found a majority of femoral bone afferent cell bodies in L3 dorsal root ganglia (DRG) that also express the sodium channel subtype Na1.8-a marker of nociceptive neurons-and lack expression of parvalbumin-a marker for proprioceptive primary afferents. Surprisingly, the response properties of bone marrow afferents to both increased intraosseous pressure and acid were unchanged by the presence of cancer. On the other hand, we found increased excitability and polymodality of cutaneous afferents innervating the ipsilateral paw in cancer bearing animals, as well as a behavioural phenotype that suggests changes at the level of the DRG contribute to secondary hypersensitivity. This study demonstrates that cutaneous afferents at distant sites from the tumour bearing tissue contribute to mechanical hypersensitivity, highlighting these cells as targets for analgesia.
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