Multipotent mesenchymal stem cells (MSCs) including bone marrow stromal cells (BMSCs) have shown analgesic efficacy in recent years. Studies suggested that the therapeutic effect of MSCs was mediated by their secreted small extracellular vesicles (sEVs) mainly exosomes. The present study evaluated the antihyperalgesic effect of BMSC-related sEVs in a mouse model of neuropathic pain involving chronic constriction injury of the infraorbital nerve (CCI-ION). Our separation protocol generated EV particles mostly sized in the range of exosomes (30-170 nm) and express exosome marker proteins CD9, CD81, and Tsg101, suggesting their endosome origin. We show that intravenous injection of BMSC-related sEVs attenuated pain hypersensitivity induced by CCI-ION as indicated by decreased mechanical hypersensitivity (von Frey test) and reduced aversion to noxious stimulation (conditioned place avoidance test). The antihyperalgesic effect of sEVs was observed in both female and male animals, and the effect was dose-dependent. sEVs from NAIVE serum-treated BMSC cultures produced short-lasting antihyperalgesia in male but not female mice, suggesting a subtle sex difference. The antihyperalgesia of sEVs from BMSC culture was blocked by the pretreatment of the culture with GM4869, the antagonist of exosome secretion, suggesting that the effect was not related to other co-isolated soluble mediators but mediated by MSC-derived exosomes. Interestingly, the prior injury condition in which sEVs were isolated favors the pain-relieving effect of sEVs. sEVs isolated from the serum of BMSC-treated animals receiving tendon ligation (TL) injury attenuated hyperalgesia for 24 h, while sEVs from the serum of BMSC-treated NAIVE animals only attenuated hyperalgesia at 3 h after injection. sEVs from the BMSC culture treated with the serum of TL rats were antihyperalgesic, but sEVs from the BMSC culture treated with the serum of naive animals were ineffective. Our results indicate that BMSC-related sEVs produced antihyperalgesia similar to that produced by BMSCs. The results suggest that the interactions between BMSCs and injury conditions are crucially important for producing efficacious sEVs/exosomes and support that the effect of sEVs could be optimized by priming BMSCs with injury-related conditions.

ABSTRACT:Chronic pain is associated with maladaptive reorganization of brain networks, particularly in the anterior cingulate cortex (ACC), contributing to the affective dimension of pain. Although peripheral capsaicin administration relieves neuropathic pain in clinics, its effects on central pain networks remain unclear. In this study, we determined the resting-state functional connectivity of ACC (ACC FC) rearrangement after infraorbital nerve chronic constriction injury (ION-CCI) and subsequent peripheral administration of capsaicin through longitudinal resting-state functional magnetic resonance imaging (fMRI) in male rats. We also conducted functional silencing of the ACC using inhibitory chemogenetic receptors to determine ACC networks commonly reversed by peripheral capsaicin and chemogenetic silencing. Infraorbital nerve chronic constriction injury produced orofacial mechanical allodynia accompanied by ACC FC changes compared to sham. A single injection of capsaicin into the maxillary skin decreased mechanical allodynia. Five days after capsaicin injection, CCI-enhanced ACC FC was significantly reduced compared to the time point before the injection in the same rats or to the rats with vehicle injection. Subsequent chemogenetic silencing of ACC in the previously vehicle-treated CCI rats reduced mechanical allodynia and suppressed CCI-enhanced ACC FC. Peripheral capsaicin and chemogenetic inhibition of ACC commonly reversed approximately one-third of the CCI-enhanced ACC FC. Affected regions included the bilateral cingulate areas, primary and secondary somatosensory cortex, primary and secondary auditory areas, hippocampus, and temporal association cortex. We conclude that peripheral capsaicin administration reverses maladaptive ACC networks in male rats with nerve injury and that peripheral nociceptors contribute to the maintenance chronic pain and peripherally targeted treatment can produce long-lasting analgesia.

Patients with myofascial pain in the head and neck region often report widespread, referred pain or secondary hypersensitivity, including headache-like. Secondary hypersensitivity originating from masticatory myalgia may result from myalgia-induced plasticity within the central nervous system, affecting referred site sensitization. The main aims of this study were to develop animal models that mimic secondary hypersensitivity and to investigate whether stimulated myalgia induces gene expression plasticity at referred sites, which may contribute to the secondary hypersensitivity phenomenon. A majority of experiments were conducted in male and female mice. Masticatory myalgia was assessed as mechanical hypersensitivity in the region over the masseter muscle (MM). Secondary hypersensitivity was evaluated by measuring mechanical hypersensitivity at sites anatomically distinct from the stimulated muscle: periorbital and MM areas. Stimulated myalgia was achieved by either a single high-dose collagenase type-II (10U; Col) or repeated low-dose Col (0.2–0.5U) injections into the MM or the temporal muscle (TM), repetitive gentle vibration applied over the MM, a single forceful mouth opening (FMO), or repeated FMO. Statistical analyses were one-way or two-way ANOVA followed by Bonferroni post-hoc tests. Stimulation of the MM, whether by single, repeated Col injections or FMO, produced inconsistent and short-lasting (1–2 days) referred pain at a periorbital area in both males and females. In contrast, stimulation of the TM using multiple paradigms reliably induced mechanical secondary hypersensitivity in two referred sites: MM and periorbital areas. MM stimulation did not exhibit sex-dependent mechanical hypersensitivity in the MM area. In contrast, TM-induced secondary hypersensitivity at both the MM and periorbital areas was sex-dependent. Secondary hypersensitivity in the MM and periorbital regions following TM stimulation was accompanied by significant gene expression plasticity in both tissues. Notably, transcriptional changes in the MM after Col injection into the TM closely resembled those observed following direct Col injection into the MM. The presented data suggest that secondary hypersensitivity from masticatory myalgia can be effectively modeled in mice through stimulation of the TM. Importantly, TM stimulation-induced transcriptomic changes at MM and dura mater may generate nociceptive signaling from these sites, thereby contributing to an input network underlying secondary hypersensitivity.

Proprioception, often described as a sixth sense to perceive body position and muscle contraction, has been increasingly implicated in the development and maintenance of chronic pain, yet the underlying mechanisms remain unclear. Here, we identify the functional expression of TRPA1 in proprioceptive mesencephalic trigeminal nucleus (MeV) neurons in a mouse model of orofacial muscle pain. TRPA1 sensitization enhances somatic secretion from MeV neurons, thereby promoting the progression and persistence of inflammatory pain by disinhibiting descending pain control from the neighboring noradrenergic locus coeruleus (LCNE) neurons. Local GABAergic neurons that provide input to LCNE neurons serve as a key downstream target of MeV somatic volume transmission, forming a critical relay that transduces signals from the MeV to the LCNE system. Thus, these findings reveal a previously unrecognized somatic volume transmission that links proprioceptive and nociceptive circuits and provides a central mechanism for persistent muscle pain.

It is well-known that children have a delay between their first production of color words and acquisition of adult-like understanding. A previous study showed that this delay could be attributed to a process of gradually converging on language-specific color word boundaries. In this study, we tested this account in a second language, Japanese. We presented 12 color samples to children and then conducted production and comprehension tasks to check whether children have adult-like understanding of color words. Our results were consistent with previous findings showing that children before acquiring adult-like understanding tend to use color words systematically as overextensions of adult meanings. These results indicate that the delay between production and adult-like understanding of color words reflects a gradual process of learning language-specific color boundaries, potentially shared across languages.

Patients with myofascial pain in the head and neck area report widespread and referred pain, including headache. Existing preclinical models fail to replicate this clinical phenotype; therefore, we aimed to develop animal models mimicking referred pain phenomenon and investigate whether referred pain leads to gene plasticity at the referred sites. We modeled masticatory myalgia by stimulation of either the masseter (MM) or temporal muscle (TM) in mice. MM and TM were stimulated with a single high-dose injection of Collagenase-type II (Col), repetitive low-dose Col injections, repetitive gentle MM stimulation, or single or repetitive forceful mouth opening. Referred pain was assessed by measuring mechanical hypersensitivity in the periorbital area (representing headache-like behavior) and another masticatory muscle. Stimulation of the MM, whether through single or repetitive Col injections or mouth opening, produced inconsistent, short-lasting (1-2 days) headache-like behavior in both males and females. In contrast, stimulation of the TM, using different paradigms, triggered mechanical hypersensitivity in both the MM and the periorbital area. Referred headache-like behavior lasted longer in females compared to males, while referred myalgia in the MM was pronouncer in males. The referred pain in the MM and periorbital areas triggered by TM stimulation was associated with significant gene plasticity in the MM and dura mater. Transcriptional changes in the MM following Col injection into the TM resembled those observed after direct MM injections. Presented data imply that referred pain modeled by TM stimulation could be accounted by nociceptive signaling from multiple local sites involved in this referred pain network.

This study investigated whether infants encode better the features of a briefly occluded object if its movements are specified simultaneously by vision and audition than if they are not (data collected: 2017-2019). Experiment 1 showed that 10-month-old infants (N = 39, 22 females, White-English) notice changes in the visual pattern on the object irrespective of the stimulation received (spatiotemporally congruent audio-visual stimulation, incongruent stimulation, or visual-only; eta p2$$ {\eta}_{\mathrm{p}}<^>2 $$ = .53). Experiment 2 (N = 72, 36 female) found similar results in 6-month-olds (Test Block 1, eta p2$$ {\eta}_{\mathrm{p}}<^>2 $$ = .13), but not 4-month-olds. Experiment 3 replicated this finding with another group of 6-month-olds (N = 42, 21 females) and showed that congruent stimulation enables infants to detect changes in object trajectory (d = 0.56) in addition to object pattern (d = 1.15), whereas incongruent stimulation hinders performance.

Human infants cannot report their experiences, limiting what we can learn about their bodily awareness. However, visual cortical responses to the body, linked to visual awareness and selective attention in adults, can be easily measured in infants and provide a promising marker of bodily awareness in early life. We presented 4- and 8-month-old infants with a flickering (7.5 Hz) video of a hand being stroked and recorded steady-state visual evoked potentials (SSVEPs). In half of the trials, the infants also received tactile stroking synchronously with visual stroking. The 8-month-old, but not the 4-month-old infants, showed a significant enhancement of SSVEP responses when they received tactile stimulation concurrent with the visually observed stroking. Follow-up experiments showed that this enhancement did not occur when the visual hand was presented in an incompatible posture with the infant’s own body or when the visual stimulus was a body-irrelevant video. Our findings provide a novel insight into the development of bodily self-awareness in the first year of life.

Recent studies have revealed asymmetries in color perception for blue vs. yellow: blue appears more achromatic, whilst yellow appears more saturated. These asymmetries may reflect a prior of the visual system to attribute blue to the lighting and yellow to the objects, because lighting from the sky and in shadows tends to be bluish. To examine when these asymmetries are acquired during development, we compared visual salience for blue/gray changes versus complementary yellow/gray changes with the same chromatic contrast, in infants aged 4-8 months. The colors were shown as patches or as object colors in two different experiments. In both cases, the color changes were presented side by side alternately with a 200 ms ISI. Looking time was measured to test for a spontaneous preference of infants for yellow/gray changes. The results showed that infants aged 6-8 months, but not 4-5 months, looked longer at yellow/gray changes in both color patches and objects images. This suggests that the infants perceived the yellow/gray changes as more salient and possibly as a change in surface rather than lighting color. We also confirmed that infants have similar detection sensitivities to blue and yellow, suggesting that the different preferences for blue/gray changes and yellow/gray changes stem from a higher-order appearance or inference about color rather than a developmental difference in peripheral processing. We used the identical paradigm to also examine whether adults perceived lower salience in the color changes along a bluish axis than along a yellowish or reddish or greenish axis. The results showed similar tendencies to the infants aged 6-8 months: adults reported lower saliences in color changes along the bluish axis than other directions. Our results establish an early developmental trajectory for blue-yellow asymmetries in infants, which may depend on experience with the natural lighting asymmetries arising from sun and sky.

Recent electrophysiological studies in primates and fMRI studies in human have indicated the existence of neurons in the visual cortex that selectively respond to hues off the cardinal axes of cone-opponent color space (intermediate colors). However, it is unclear when and how the hue selectivity develops in the early developmental stage of the visual system. In the present study, we measured hue selectivity of brain activity in infants using steady-state visual evoked potentials (SSVEPs). Infants observed the reversals of a checkerboard pattern during the measurement. Half of the tiles of checkerboard were filled with the background hue (Equal Energy White) and the rest of them were filled with a test hue. Twelve test hues were chosen from a hue circle, which had a color contrast of ∆L = 8 % and ∆S = 80 % with respect to the background color, in an equiluminant plane defined by the cone-opponent color space (Macleod & Boynton, 1979). To yield SSVEP responses, the pattern flipped at 5 Hz during a 5 seconds testing trial. The test hue changed every second along the hue circle during the measurement. Fourteen 5-6-months old infants participated in the study. The SSVEP response amplitudes in infants were assessed by a model that assumes that the responses of intermediate hues are the sum of responses of two nearby cardinal mechanisms. Our results revealed that infant’s SSVEP responses were more likely to be dominated by cone-opponent representation, in contrast to adults’ SSVEP responses in which amplitudes were significantly larger in intermediate hues (magenta and lime-green; Kaneko, Kuriki & Andersen, 2020). Meanwhile, average SSVEP amplitude showed a biased response along the red/green cardinal axis, which is also different from adults. This bias could result from the faster development of red/green cardinal mechanism (Teller, 1998).

Perceiving one’s own body underpins skilled interactions with the external world and plays a fundamental role in the sense of self. Findings across experimental psychology and neuroscience show that body perception depends on integrating bodily information across multiple senses. However, the emergence of such multisensory abilities in early human development is just starting to be investigated. It is now generally established that human infants are sensitive to the spatiotemporal congruency between cues about the body coming from different senses, even with only a few months or even days of postnatal experience. Conversely, other abilities appear to have a more protracted development, such as the ability to make the crossmodal links required to locate tactile stimuli in external space, i.e., the “remapping problem” (Driver & Spence, 1998; Heed, Buchholz, Engel, & Röder, 2015), which develops gradually in the first year of life. This article briefly reviews the scientific literature concerning body representations in early infancy, highlighting the important role of visual experience in the development of these fundamental representational abilities.

Oxytocin is a well-known neurohypophysial hormone that plays an important role in behavioral anxiety and nociception. Two major forms of long-term potentiation, presynaptic LTP (pre-LTP) and postsynaptic LTP (post-LTP), have been characterized in the anterior cingulate cortex (ACC). Both pre-LTP and post-LTP contribute to chronic-pain-related anxiety and behavioral sensitization. The roles of oxytocin in the ACC have not been studied. Here, we find that microinjections of oxytocin into the ACC attenuate nociceptive responses and anxiety-like behavioral responses in animals with neuropathic pain. Application of oxytocin selectively blocks the maintenance of pre-LTP but not post-LTP. In addition, oxytocin enhances inhibitory transmission and excites ACC interneurons. Similar results are obtained by using selective optical stimulation of oxytocin-containing projecting terminals in the ACC in animals with neuropathic pain. Our results demonstrate that oxytocin acts on central synapses and reduces chronic-pain-induced anxiety by reducing pre-LTP.

我々が日常に体験している複雑な視知覚世界は，どのように形成されるのだろうか。その発達プロセスを解明するため，講演者はこれまで，乳児を対象とした実験を行ってきた。本講演では，乳児におけるカテゴリカル色知覚と前恒常性を中心に報告する。

Temporomandibular disorder (TMD) is commonly comorbid with fibromyalgia syndrome (FMS). The incidence of these pain conditions is prevalent in women and prone to mental stress. Chronic pain symptoms in patients with FMS and myofascial TMD (mTMD) are severe and debilitating. In the present study, we developed a new animal model to mimic the comorbidity of TMD and FMS. In ovariectomized female rats, repeated forced swim (FS) stress induced mechanical allodynia and thermal hyperalgesia in the hindpaws of the 17β-estradiol (E2) treated rats with orofacial inflammation. Subcutaneous injection of E2, injection of complete Freund’s adjuvant (CFA) into masseter muscles or FS alone did not induce somatic hyperalgesia. We also found that the somatic hyperalgesia was accompanied by upregulation of GluN1 receptor and serotonin (5-hydroxytryptamine, 5-HT)3A receptor expression in the dorsal horn of spinal cord at L4-L5 segments. Intrathecal injection of N-methyl-D-aspartic acid receptor (NMDAR) antagonist 2-amino-5-phosphonovaleric acid (APV) or 5-HT3 receptor antagonist Y-25130 blocked stress-induced wide-spreading hyperalgesia. These results suggest that NMDAR-dependent central sensitization in the spinal dorsal horn and 5-HT-dependent descending facilitation contribute to the development of wide-spreading hyperalgesia in this comorbid pain model.

Craniofacial muscle pain is highly prevalent in temporomandibular disorders but is difficult to treat. Enhanced understanding of neurobiology unique to craniofacial muscle pain should lead to the development of novel mechanism-based treatments. Herein, we review recent studies to summarize neural pathways of craniofacial muscle pain. Nociceptive afferents in craniofacial muscles are predominantly peptidergic afferents enriched with TRPV1. Signals from peripheral glutamate receptors converge onto TRPV1, leading to mechanical hyperalgesia. Further studies are needed to clarify whether hyperalgesic priming in nonpeptidergic afferents or repeated acid injections also affect craniofacial muscle pain. Within trigeminal ganglia, afferents innervating craniofacial muscles interact with surrounding satellite glia, which enhances the sensitivity of the inflamed neurons as well as nearby uninjured afferents, resulting in hyperalgesia and ectopic pain originating from adjacent orofacial tissues. Craniofacial muscle afferents project to a wide area within the trigeminal nucleus complex, and central sensitization of medullary dorsal horn neurons is a critical factor in muscle hyperalgesia related to ectopic pain and emotional stress. Second-order neurons project rostrally to pathways associated with affective pain, such as parabrachial nucleus and medial thalamic nucleus, as well as sensory-discriminative pain, such as ventral posteromedial thalamic nuclei. Abnormal endogenous pain modulation can also contribute to chronic muscle pain. Descending serotonergic circuits from the rostral ventromedial medulla facilitate activation of second-order neurons in the trigeminal nucleus complex, which leads to the maintenance of mechanical hyperalgesia of inflamed masseter muscle. Patients with temporomandibular disorders exhibit altered brain networks in widespread cortical and subcortical regions. Recent development of methods for neural circuit manipulation allows silencing of specific hyperactive neural circuits. Chemogenetic silencing of TRPV1-expressing afferents or rostral ventromedial medulla neurons attenuates hyperalgesia during masseter inflammation. It is likely, therefore, that further delineation of neural circuits mediating craniofacial muscle hyperalgesia potentially enhances treatment of chronic muscle pain conditions.

Trigeminal neuropathic pain (TNP) is often resistant to current pharmacotherapy, and there is a pressing need to develop more efficacious treatments. Capsaicin is a pungent ingredient of chili peppers and specifically activates transient receptor potential vanilloid subtype 1 (TRPV1), a Ca2+-permeable ion channel. Topical capsaicin invariably induces burning pain. Paradoxically, the transient pain is often followed by prolonged attenuation of the preexisting pathologic pain from the same region. However, the mechanisms underlying capsaicin-induced analgesia are not well understood. Although the reports of the involvement of TRPV1 and TRPV1+ afferents in neuropathic pain are controversial, we recently demonstrated that TRPV1 and TRPV1+ afferents are involved in mechanical hyperalgesia in mice with chronic constriction injury of the infraorbital nerve (ION-CCI). Consistently, chemogenetic inhibition of TRPV1-lineage (TRPV1-LN) afferents attenuated mechanical hyperalgesia and ongoing pain. In mice with ION-CCI, we found that a single focal injection of capsaicin into facial skin led to attenuation of mechanical hyperalgesia over two weeks. Capsaicin treatment also attenuated secondary hyperalgesia in extraterritorial mandibular skin. Furthermore, capsaicin treatment decreased ongoing pain. Longitudinal in vivo two-photon imaging of cutaneous nerve fibers showed that such capsaicin-induced analgesia is correlated with cutaneous nerve terminal density. Furthermore, preventing capsaicin-induced ablation of afferent terminals by co-administration of capsaicin with MDL28170, an inhibitor of calpain, abolished capsaicin-induced analgesia. These results suggest that a single focal injection of capsaicin induces long-lasting analgesia for neuropathic pain via selective ablation of TRPV1+ afferent terminals and that TRPV1+ afferents contribute to the maintenance of TNP.

Body representations are products of complex multisensory interactions and are closely related to motor control and the sense of self. A number of claims have been made that humans are born with an innate ability to perceive their own bodies (Rochat, 2010), with some evidence of visual tactile interactions present in the early months of life (Filippetti et al., 2013; Freier et al., 2016). However, only a little is known about how the multisensory body representations develop. In the present study, we used the steady-state visually evoked potentials (SSVEP) to investigate the development of tactile-visual cortical interactions underlying body representations in infants. In Experiment 1, twelve 4-month-old and twelve 8-month-old infants watched a visual presentation in which a hand was stroked with a metal tube. To elicit the SSVEP, the video flashed at 7.5 Hz. In the tactile-visual condition the infant’s own hand was also stroked by a tube whilst they watched the movie. In the vision-only condition, no tactile stimulus was applied to the infant’s hand. We found larger SSVEPs in the tactile-visual condition than the vision-only condition in 8-month-old infants, but no difference between the two conditions in the 4-month-olds. In Experiment 2, we presented an irrelevant video to 8-month-old infants rather than a hand. The enhancement of tactile stimuli on SSVEP was absent in this case, demonstrating that there was some degree of body-specific information was required to drive the tactile enhancements of visual cortical processing seen in Experiment 1. Taken together, our results indicate that tactile influences on visual processing of bodily information develops between 4 and 8 months of age.

Perceiving and judging food quality is indispensable in daily life. The present study examined this ability's development in infants during the early postnatal months. We tested if infants aged 5-8 months can discriminate different degree of freshness in cabbage, strawberry, carrot, and spinach. In Experiment 1, images of fresh and degraded vegetables were presented side by side; infants aged 7-8 months significantly preferred fresh over degraded cabbage images. In Experiments 2 and 3, infants aged 7-8 months maintained their preference when the images were achromatic, but no longer preferred the fresh cabbage images when pixels in those images were randomized. Given these results, we suggest that the ability to discriminate different degrees of freshness, at least for cabbage, develops at approximately 7-8 months of age, which is the time probably prior to taste learning.