Identification of Proteolysis Targeting Chimeras (PROTACs) for Lysine Demethylase 5 and Their Neurite Outgrowth-Promoting Activity.

Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer's disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.

Region-specific activation in the accumbens nucleus by itch with modified scratch efficacy in mice - a model-free multivariate analysis.

Itch is a protective/defensive function with divalent motivational drives. Itch itself elicits an unpleasant experience, which triggers the urge to scratch, relieving the itchiness. Still, it can also result in dissatisfaction when the scratch is too intense and painful or unsatisfactory due to insufficient scratch effect. Therefore, it is likely that the balance between the unpleasantness/pleasure and satisfaction/unsatisfaction associated with itch sensation and scratching behavior is determined by complex brain mechanisms. The physiological/pathological mechanisms underlying this balance remain largely elusive. To address this issue, we targeted the "reward center" of the brain, the nucleus accumbens (NAc), in which itch-responsive neurons have been found in rodents. We examined how neurons in the NAc are activated or suppressed during histamine-induced scratching behaviors in mice. The mice received an intradermal injection of histamine or saline at the neck, and the scratching number was analyzed by recording the movement of the bilateral hind limbs for about 45 min after injection. To experimentally manipulate the scratch efficacy in these histamine models, we compared histamine's behavioral and neuronal effects between mice with intact and clipped nails on the hind paws. As expected, the clipping of the hind limb nail increased the number of scratches after the histamine injection. In the brains of mice exhibiting scratching behaviors, we analyzed the expression of the c-fos gene (Fos) as a readout of an immediate activation of neurons during itch/scratch and dopamine receptors (Drd1 and Drd2) using multiplex single-molecule fluorescence in situ hybridization (RNAscope) in the NAc and surrounding structures. We performed a model-free analysis of gene expression in geometrically divided NAc subregions without assuming the conventional core-shell divisions. The results indicated that even within the NAc, multiple subregions responded differentially to various itch/scratch conditions. We also found different clusters with neurons showing similar or opposite changes in Fos expression and the correlation between scratch number and Fos expression in different itch/scratch conditions. These regional differences and clusters would provide a basis for the complex role of the NAc and surrounding structures in encoding the outcomes of scratching behavior and itchy sensations.

Identification of a novel histone H2A mono-ubiquitination-inhibiting cell-active small molecule.

Histone H2A mono-ubiquitination plays important roles in epigenetic gene expression and is also involved in tumorigenesis. Small molecules controlling H2A ubiquitination are of interest as potential chemical tools and anticancer drugs. To identify novel small molecule inhibitors of H2A ubiquitination, we synthesized and evaluated several compounds designed based on PRT4165 (1), which is a reported histone ubiquitin ligase RING1A inhibitor. We found that compound 11b strongly inhibited the viability and reduced histone H2A mono-ubiquitination in human osteosarcoma U2OS cells. Therefore, compound 11b is a promising lead compound for the development of H2A histone ubiquitination-inhibiting small molecules.

Presynaptic inhibition of excitatory synaptic transmission from the calcitonin gene-related peptide-containing parabrachial neurons to the central amygdala in mice - unexpected influence of systemic inflammation thereon.

The monosynaptic connection from the lateral parabrachial nucleus (LPB) to the central amygdala (CeA) serves as a fundamental pathway for transmitting nociceptive signals to the brain. The LPB receives nociceptive information from the dorsal horn and spinal trigeminal nucleus and sends it to the "nociceptive" CeA, which modulates pain-associated emotions and nociceptive sensitivity. To elucidate the role of densely expressed mu-opioid receptors (MORs) within this pathway, we investigated the effects of exogenously applied opioids on LPB-CeA synaptic transmission, employing optogenetics in mice expressing channelrhodopsin-2 in LPB neurons with calcitonin gene-related peptide (CGRP). A MOR agonist ([D-Ala2,N-Me-Phe4,Glycinol5]-enkephalin, DAMGO) significantly reduced the amplitude of light-evoked excitatory postsynaptic currents (leEPSCs), in a manner negatively correlated with an increase in the paired-pulse ratio. An antagonist of MORs significantly attenuated these effects. Notably, this antagonist significantly increased leEPSC amplitude when applied alone, an effect further amplified in mice subjected to lipopolysaccharide injection 2 h before brain isolation, yet not observed at the 24-h mark. We conclude that opioids could shut off the ascending nociceptive signal at the LPB-CeA synapse through presynaptic mechanisms. Moreover, this gating process might be modulated by endogenous opioids, and the innate immune system influences this modulation.

Pregabalin attenuates long-lasting post-inflammatory nociplastic mechanical sensitization in mice.

Nociplastic pain, the most recently proposed mechanistic descriptor of chronic pain, is the pain resulting from an altered nociceptive system and network without clear evidence of nociceptor activation, injury or disease in the somatosensory system. As the pain-associated symptoms in many patients suffering from undiagnosed pain would result from the nociplastic mechanisms, it is an urgent issue to develop pharmaceutical therapies that would mitigate the aberrant nociception in nociplastic pain. We have recently reported that a single injection of formalin to the upper lip shows sustained sensitization lasting for more than 12 days at the bilateral hindpaws, where there is no injury or neuropathy in rats. Using the equivalent model in mice, we show that pregabalin (PGB), a drug used for treating neuropathic pain, significantly attenuates this formalin-induced widespread sensitization at the bilateral hindpaws, even on the 6 day after the initial single orofacial injection of formalin. On the 10th day after formalin injection, the hindlimb sensitization before PGB injection was no more significant in mice receiving daily PGB injections, unlike those receiving daily vehicle injections. This result suggests that PGB would act on the central pain mechanisms that undergo nociplastic changes triggered by initial inflammation and mitigate widespread sensitization resulting from the established changes.

Inflammation and subsequent nociceptor sensitization in the bone marrow are involved in an animal model of osteoarthritis pain.

AIMS: This study aimed to determine whether pathological changes in the bone marrow cause Osteoarthritis (OA) pain based on magnetic resonance imaging (MRI), immunohistochemistry, and electrophysiology. MAIN METHODS: Adjuvant-induced arthritis (AIA) was achieved by injecting 150 µL of complete Freund's adjuvant into the right knee joints of male Sprague-Dawley rats. AIA rats were compared with saline-injected rats. KEY FINDINGS: AIA significantly induced mechanical hyperalgesia and spontaneous pain in the right hind paw 1-14 days after induction. Intratibial injection of 50 µL of 1 % lidocaine significantly suppressed AIA-induced mechanical hyperalgesia (p = 0.0001) and spontaneous pain (p = 0.0006) 3 days after induction. In T2-weighted MRI, AIA induced high-signal intensity within the proximal tibial metaphysis, and the mean T2 values in this area significantly increased on days 3 (p = 0.0043) and 14 (p = 0.0012) after induction. AIA induced intraosseous edema and significantly increased the number of intraosseous granulocytes on days 3 (p < 0.0001) and 14 (p < 0.0001) after induction. The electrophysiological study on days 3-7 after induction showed significantly increased spontaneous firing rates (p = 0.0166) and evoked responses to cutaneous stimuli (brush, p < 0.0001; pinching, p = 0.0359) in the right hind paw plantar surface and intratibial stimuli (p = 0.0002) in wide-dynamic-range neurons of the spinal dorsal horn. SIGNIFICANCE: Intraosseous changes caused by OA induce hypersensitivity in the sensory afferents innervating bone marrow may be involved in OA pain. Novel bone marrow-targeted therapies could be beneficial for treating OA pain.

Delivery-Dependent Shift in Oxytocin-Responsive Cell Population in the Central Amygdala of the Female Rat.

INTRODUCTION: Despite its recent reputation as prosocial neurohormone, the most important physiological role of oxytocin (OT) is stimulating uterine contractions. Though it is well known that plasma OT concentrations change drastically during delivery, it remains unexplored whether and how OT receptors in the maternal brain are activated. We examined whether the responses of cells in the central amygdala (CeA), an OT receptor-rich limbic site involved in pain and fear memory regulation, to exogenously applied OT analogue, Thr-Gly-OT (TGOT), vary depending on delivery. METHODS: Intracellular Ca2+ dynamics of the CeA cells were visualized in brain slices from female rats at virgin (VG), during pregnancy term (PT) days 16-21, within 24 h after delivery (G0), and within 1-3 days after delivery (G3). The Ca2+ responses to 1 µM TGOT, 20 mM KCl (high K), and 300 µM ADP were compared. RESULTS: We found that fraction of cells responding to TGOT, high K, and ADP differed significantly between the four delivery-associated terms. In particular, the fraction of cells responding to TGOT (TGOT responders) significantly increased from VG and PT at G0 and G3. Furthermore, the significant positive correlation between TGOT and high K response in TGOT and high K responders was reduced at G0, while that between TGOT and ADP responses in TGOT and ADP responders was increased at G0. CONCLUSION: These results indicate that the responses of CeA cells to an OT receptor agonist markedly change around delivery, which might play a role in controlling the labor-related pain and post-delivery emotional complications.

Selective degradation of histone deacetylase 8 mediated by a proteolysis targeting chimera (PROTAC).

We developed a first-in-class proteolysis targeting chimera (PROTAC) for selective degradation of histone deacetylase 8 (HDAC8). The PROTAC induced degradation of HDAC8 without affecting the levels of other HDACs in cellular assays, and inhibited the growth of T-cell leukemia Jurkat cells more potently than a conventional HDAC8 inhibitor.

Acetaminophen and pregabalin attenuate central sensitization in rodent models of nociplastic widespread pain.

The "nociplastic pain," a recently proposed novel mechanistic pain descriptor, is defined as pain occurring through altered nociception without nociceptor activation and nerve injury. Nociplastic pain is often characterized by widespread pain sensitization (WSP) in multiple body regions (Fitzcharles et al., 2021). As many patients with primary chronic pain would have nociplastic backgrounds, developing appropriate methods to evaluate drug effects against nociplastic pain in animal model is in great demand. Using two rat models with the WSP involving central amygdala (CeA) activation by orofacial inflammation or direct chemogenetic activation (Sugimoto et al., 2021), we examined whether widely used analgesics, acetaminophen (AcAph), pregabalin (PGB), and duloxetine (DLX) could attenuate the WSP. AcAph (100 or 200 mg/kg, i.p.) significantly elevated 50%-paw withdrawal threshold (PWT50), which had been lowered significantly by upper lip injection of formalin, or systemic injection of clozapine-N-oxide in the rats with excitatory designer receptors (hM3Dq) expressed in the right CeA. This effect lasted for > ∼4 h. PGB (30 mg/kg, i.p.) also significantly counteracted the lowered PWT50 in rats with orofacial formalin injection for >∼6 h. DLX was ineffective on the WSP. Based on these results, we propose that these preclinical models could be used to evaluate drug effects for primary chronic pain. We conclude that the widely used pain killers, AcAph and PGB, also relieve nociplastic widespread sensitization in the absence of ongoing nociceptor activation and nerve injury.

Attenuation of widespread hypersensitivity to noxious mechanical stimuli by inhibition of GABAergic neurons of the right amygdala in a rat model of chronic back pain.

BACKGROUND: Chronic primary low back pain may be associated with hyperalgesia in uninjured tissues and with decreased pain inhibition. Previous studies have shown that the amygdala is involved in pain regulation and chronic pain, that neuronal activity in the amygdala is altered in models of persistent pain, and that the central nucleus of the right amygdala plays an active role in widespread hypersensitivity to noxious stimuli. METHODS: Behavioral, electrophysiological, biochemical, and chemogenetic methods were used to examine the role of the central nucleus of the right amygdala in hypersensitivity to noxious stimuli in a rat model of chronic back pain induced by a local injection of Complete Freund Adjuvant (CFA) in paraspinal muscles. RESULTS: CFA produced chronic inflammation limited to the injected area. CFA-treated rats showed increased pain-like (liking) behaviors during the formalin test compared with controls. They also showed widespread mechanical hypersensitivity compared with controls, which persisted for 2 months. This widespread hypersensitivity was accompanied by altered activity of different types of right amygdala neurons, as shown by extracellular recordings. Plasmatic levels of IL-1ß, IL-6, and TNF-α were not elevated after 1 or 2 months, indicating that persistent widespread hypersensitivity is not caused by persistent systemic inflammation. However, chemogenetic inhibition of GABAergic neurons in the right amygdala attenuated widespread mechanical hypersensitivity. CONCLUSIONS: These findings indicate that chronic widespread mechanical hypersensitivity in a model of chronic back pain can be attenuated by inhibiting GABAergic neurons of the right amygdala, and that widespread hypersensitivity is not maintained by chronic systemic inflammation. SIGNIFICANCE: The amygdala is a key structure involved in pain perception and modulation. The present results indicate that the GABAergic neurons of its central nucleus are involved in widespread hypersensitivity to noxious stimuli in a rat model of chronic back pain. The inhibition of amygdala GABAergic neurons may be a potential target for future interventions in patients with chronic back pain.

Input-dependent synaptic suppression by pregabalin in the central amygdala in male mice with inflammatory pain.

Pregabalin (PGB) is a synthetic amino acid compound most widely prescribed for chronic peripheral and central neuropathic pain. PGB is a ligand for the α2δ1 subunit of voltage-dependent calcium channels, and its binding reduces neurotransmitter release and thus inhibits synaptic transmission. The central nucleus of the amygdala (CeA) is a kernel site for the enhanced nociception-emotion link in chronic pain. The nociceptive information is conveyed to the CeA via the following two pathways: 1) the pathway arising from the basolateral amygdala (BLA), which carries nociceptive information mediated by the thalamocortical system, and 2) that arising from the external part of the pontine lateral parabrachial nucleus (LPB), that forms the final route of the spino-parabrachio-amygdaloid pathway that conveys nociceptive information directly from the superficial layer of the spinal dorsal horn. We compared the effects of PGB on the excitatory postsynaptic currents of neurons in the right CeA in response to electrical stimulation of BLA and LPB pathways using the whole-cell patch-clamp technique. Inflammatory pain was induced by intraplantar injection of formalin solution at the left hind paw. At eight hours post-formalin, PGB reduced EPSCs amplitude of the BLA-to-CeA synaptic transmission, accompanied by a significant increase in the PPR, suggesting a decreased release probability from the presynaptic terminals. In addition, these effects of PGB were only seen in inflammatory conditions. PGB did not affect the synaptic transmission at the LPB-to-CeA pathway, even in formalin-treated mice. These results suggest PGB improves not simply the aberrantly enhanced nociception but also various pain-associated cognitive and affective consequences in patients with chronic nociplastic pain.

Identification of Potent and Selective Inhibitors of Fat Mass Obesity-Associated Protein Using a Fragment-Merging Approach.

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

Sustained microglial activation in the area postrema of collagen-induced arthritis mice.

BACKGROUND: Central nervous system (CNS)-mediated symptoms, such as fatigue, depression, and hyperalgesia, are common complications among patients with rheumatoid arthritis (RA). However, it remains unclear how the peripheral pathology of RA spreads to the brain. Accumulated evidence showing an association between serum cytokine levels and aberrant CNS function suggests that humoral factors participate in this mechanism. In contrast to the well-known early responses of microglia (CNS-resident immune cells) in the area postrema [AP; a brain region lacking a blood-brain barrier (BBB)] to experimental inflammation, microglial alterations in the AP during chronic inflammation like RA remain unclear. Therefore, to determine whether microglia in the AP can react to persistent autoimmune-arthritis conditions, we analyzed these cells in a mouse model of collagen-induced arthritis (CIA). METHODS: Microglial number and morphology were analyzed in the AP of CIA and control mice (administered Freund's adjuvant or saline). Immunostaining for ionized calcium-binding adaptor molecule-1 was performed at various disease phases: "pre-onset" [post-immunization day (PID) 21], "establishment" (PID 35), and "chronic" (PID 56 and 84). Quantitative analyses of microglial number and morphology were performed, with principal component analysis used to classify microglia. Interleukin-1ß (IL-1ß) mRNA expression was analyzed by multiple fluorescent in situ hybridization and real-time polymerase chain reaction. Behavioral changes were assessed by sucrose preference test. RESULTS: Microglia in the AP significantly increased in density and exhibited changes in morphology during the establishment and chronic phases, but not the pre-onset phase. Non-subjective clustering classification of cell morphology (CIA, 1,256 cells; saline, 852 cells) showed that the proportion of highly activated microglia increased in the CIA group during establishment and chronic phases. Moreover, the density of IL-1ß-positive microglia, a hallmark of functional activation, was increased in the AP. Sucrose preferences in CIA mice negatively correlated with IL-1ß expression in brain regions containing the AP. CONCLUSIONS: Our findings demonstrate that microglia in the AP can sustain their activated state during persistent autoimmune arthritis, which suggests that chronic inflammation, such as RA, may affect microglia in brain regions lacking a BBB and have various neural consequences.

Active role of the central amygdala in widespread mechanical sensitization in rats with facial inflammatory pain.

ABSTRACT: Widespread or ectopic sensitization is a hallmark symptom of chronic pain, characterized by aberrantly enhanced pain sensitivity in multiple body regions remote from the site of original injury or inflammation. The central mechanism underlying widespread sensitization remains unidentified. The central nucleus of the amygdala (also called the central amygdala, CeA) is well situated for this role because it receives nociceptive information from diverse body sites and modulates pain sensitivity in various body regions. In this study, we examined the role of the CeA in a novel model of ectopic sensitization of rats. Injection of formalin into the left upper lip resulted in latent bilateral sensitization in the hind paw lasting >13 days in male Wistar rats. Chemogenetic inhibition of gamma-aminobutyric acid-ergic neurons or blockade of calcitonin gene-related peptide receptors in the right CeA, but not in the left, significantly attenuated this sensitization. Furthermore, chemogenetic excitation of gamma-aminobutyric acid-ergic neurons in the right CeA induced de novo bilateral hind paw sensitization in the rats without inflammation. These results indicate that the CeA neuronal activity determines hind paw tactile sensitivity in rats with remote inflammatory pain. They also suggest that the hind paw sensitization used in a large number of preclinical studies might not be simply a sign of the pain at the site of injury but rather a representation of the augmented CeA activity resulting from inflammation/pain in any part of the body or from activities of other brain regions, which has an active role of promoting defensive/protective behaviors to avoid further bodily damage.

Mole-Ratio-Dependent Reversible Transformation between 2:2 and Cyclic 3:6 Silver(I) Complexes with an Argentivorous Molecule.

A tetra-armed cyclen (L) with two substituted 3,5-difluorobenzyl and two substituted pyridine-4-yl methyl groups at the 1,4- and 7,10-positions of the cyclen ring as side arms was synthesized. When L was reacted with 1 equiv of the silver(I), dimetallo[3.3]paracyclophane-like 2:2 cyclic dimer, [Ag2(L)2](PF6)2, was obtained. The reaction of L with 2 equiv of silver(I) gave a 3:6 cyclic trimer, [Ag6(L)3(CH3CN)3](OTf)6·3CH3CN. Furthermore, reversible complexation between the 2:2 cyclic dimer and 3:6 cyclic trimer was confirmed by 1H NMR and the CSI mass in the addition of silver(I) or the [2.2.2]cryptand.

Design, Synthesis, and Biological Evaluation of Lysine Demethylase 5†C Degraders.

Lysine demethylase 5 C (KDM5C) controls epigenetic gene expression and is attracting great interest in the field of chemical epigenetics. KDM5C has emerged as a therapeutic target for anti-prostate cancer agents, and recently we identified triazole 1 as an inhibitor of KDM5C. Compound 1 exhibited highly potent KDM5C-inhibitory activity in in vitro enzyme assays, but did not show strong anticancer effects. Therefore, a different approach is needed for the development of anticancer agents targeting KDM5C. Here, we attempted to identify KDM5C degraders by focusing on a protein-knockdown strategy. Compound 3 b, which was designed based on compound 1, degraded KDM5C and inhibited the growth of prostate cancer PC-3 cells more strongly than compound 1. These findings suggest that KDM5C degraders are more effective as anticancer agents than compounds that only inhibit the catalytic activity of KDM5C.

Discovery of gamma-mangostin from Garcinia mangostana as a potent and selective natural SIRT2 inhibitor.

Studies have suggested that sirtuin inhibition may have beneficial effects on several age-related diseases such as neurodegenerative disorders and cancer. Garcinia mangostana is a well-known tropical plant found mostly in South East Asia with several positive health effects. Some of its phytochemicals such as α-mangostin was found to be able to modulate sirtuin activity in mice and was implicated with inflammation, diabetes and obesity. However, comprehensive studies on sirtuin activity by the prenylated xanthones extracted from Garcinia mangostana have yet to be reported. The present study led to the discovery and identification of γ-mangostin as a potent and selective SIRT2 inhibitor. It was demonstrated that γ-mangostin was able to increase the α-tubulin acetylation in MDA-MD-231 and MCF-7 breast cancer cells. It was also found to possess potent antiproliferative activity against both cell lines. In addition, it was able to induce neurite outgrowth in the N2a cells.

Alterations of voluntary behavior in the course of disease progress and pharmacotherapy in mice with collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis and bone destruction at the joints, causing pain and motor disturbance. Despite the better control of inflammation and joint deformity afforded by modern disease-modifying anti-rheumatic drugs, many patients with RA remain dissatisfied with their treatment, primarily because of sensory-emotional distress. Pre-clinical tests that can evaluate not only the symptoms of arthritis but also the associated pain as sensory-emotional experience are urgently needed. METHODS: Here, we introduce two types of novel methods for evaluation of voluntary behavior in a commonly used model of RA (collagen-induced arthritis; CIA) in male mice. First, spontaneous motor activity was assessed with a running wheel placed in home cages and the number of rotations was continuously recorded in a 12:12-h light environment. Second, temperature preference was assessed by measuring the time spent in either of the floor plates with augmenting (25 to 49 °C) or fixed temperature (25 °C). We also evaluated the effects of tofacitinib on CIA-associated changes in voluntary wheel running and temperature preference. RESULTS: We detected a significant decrease in voluntary wheel running, a significant shift in the distribution of movement in the dark phase, and a significant increase in the time spent in warmer environments than the room temperature in the mice with CIA. These alterations in voluntary behavior have never been described with conventional methods. We also revealed tofacitinib-resistant significant changes in the voluntary behavior and choice of temperature despite significant mitigation of the symptoms of arthritis. CONCLUSIONS: We described for the first time significant alterations of the voluntary behavior of the mice with CIA during the clinical periods, indicating that the overall physical/motivational states and its circadian variation, as well as the specific preference to a certain environmental temperature, are modified in the mice with CIA, as observed in human patients. Some of these did not parallel with the conventional arthritis scores, particularly during the pharmacotherapy suggesting that mice with CIA show not only the peripheral symptoms but also the central consequences. The use of these approaches would also help clarify the biological mechanisms underlying physician-patient discordance in the assessment of RA.

Primary Role of the Amygdala in Spontaneous Inflammatory Pain- Associated Activation of Pain Networks - A Chemogenetic Manganese-Enhanced MRI Approach.

Chronic pain is a major health problem, affecting 10-30% of the population in developed countries. While chronic pain is defined as "a persistent complaint of pain lasting for more than the usual period for recovery," recently accumulated lines of evidence based on human brain imaging have revealed that chronic pain is not simply a sustained state of nociception, but rather an allostatic state established through gradually progressing plastic changes in the central nervous system. To visualize the brain activity associated with spontaneously occurring pain during the shift from acute to chronic pain under anesthetic-free conditions, we used manganese-enhanced magnetic resonance imaging (MEMRI) with a 9.4-T scanner to visualize neural activity-dependent accumulation of manganese in the brains of mice with hind paw inflammation. Time-differential analysis between 2- and 6-h after formalin injection to the left hind paw revealed a significantly increased MEMRI signal in various brain areas, including the right insular cortex, right nucleus accumbens, right globus pallidus, bilateral caudate putamen, right primary/secondary somatosensory cortex, bilateral thalamus, right amygdala, bilateral substantial nigra, and left ventral tegmental area. To analyze the role of the right amygdala in these post-formalin MEMRI signals, we repeatedly inhibited right amygdala neurons during this 2-6-h period using the "designer receptors exclusively activated by designer drugs" (DREADD) technique. Pharmacological activation of inhibitory DREADDs expressed in the right amygdala significantly attenuated MEMRI signals in the bilateral infralimbic cortex, bilateral nucleus accumbens, bilateral caudate putamen, right globus pallidus, bilateral ventral tegmental area, and bilateral substantia nigra, suggesting that the inflammatory pain-associated activation of these structures depends on the activity of the right amygdala and DREADD-expressing adjacent structures. In summary, the combined use of DREADD and MEMRI is a promising approach for revealing regions associated with spontaneous pain-associated brain activities and their causal relationships.

Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity.

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.