Three weeks of exercise therapy altered brain functional connectivity in fibromyalgia inpatients.

Background: Fibromyalgia (FM) is a chronic pain syndrome characterized by widespread pain, tenderness, and fatigue. Patients with FM have no effective medication so far, and their activity of daily living and quality of life are remarkably impaired. Therefore, new therapeutic approaches are awaited. Recently, exercise therapy has been gathering much attention as a promising treatment for FM. However, the underlying mechanisms are not fully understood, particularly, in the central nervous system, including the brain. Therefore, we investigated functional connectivity changes and their relationship with clinical improvement in patients with FM after exercise therapy to investigate the underlying mechanisms in the brain using resting-state fMRI (rs-fMRI) and functional connectivity (FC) analysis. Methods: Seventeen patients with FM participated in this study. They underwent a 3-week exercise therapy on in-patient basis and a 5-min rs-fMRI scan before and after the exercise therapy. We compared the FC strength of sensorimotor regions and the mesocortico-limbic system between two scans. We also performed a multiple regression analysis to examine the relationship between pre-post differences in FC strength and improvement of patients' clinical symptoms or motor abilities. Results: Patients with FM showed significant improvement in clinical symptoms and motor abilities. They also showed a significant pre-post difference in FC of the anterior cingulate cortex and a significant correlation between pre-post FC changes and improvement of clinical symptoms and motor abilities. Although sensorimotor regions tended to be related to the improvement of general disease severity and depression, brain regions belonging to the mesocortico-limbic system tended to be related to the improvement of motor abilities. Conclusion: Our 3-week exercise therapy could ameliorate clinical symptoms and motor abilities of patients with FM, and lead to FC changes in sensorimotor regions and brain regions belonging to the mesocortico-limbic system. Furthermore, these changes were related to improvement of clinical symptoms and motor abilities. Our findings suggest that, as predicted by previous animal studies, spontaneous brain activities modified by exercise therapy, including the mesocortico-limbic system, improve clinical symptoms in patients with FM.

Exercise-induced increase in M2 macrophages accelerates wound healing in young mice.

Moderate-intensity exercise performed during wound healing has been reported to decrease inflammatory cytokines and chemokines and accelerate wound healing. However, its effect on macrophage phenotype and the mechanism by which exercise accelerates wound healing remain unclear. The purpose of this study was to investigate the effect of exercise on macrophage phenotype during wound healing and to clarify the relationship between angiogenesis and wound healing. 12-week-old male C57BL/6J mice were divided into sedentary (n = 6) and exercise groups (n = 6). The exercise group performed moderate-intensity treadmill running exercise (9.0 m/min, 60 min) for 10 days. Double immunofluorescence analysis was performed using F4/80+ inducible nitric oxide synthase (iNOS)+ for M1 macrophages, F4/80+ transforming growth factor-beta (TGF-ß)1+ for M2 macrophages, and CD31+ alpha smooth muscle actin (α-SMA)+ for angiogenesis. The exercise group showed significantly accelerated wound healing compared with the sedentary group. From early wound healing onward, exercise significantly inhibited M1 macrophage infiltration and increased M2 macrophage count. Exercise also significantly increased angiogenesis. Furthermore, the M2 macrophage phenotype was significantly correlated with angiogenesis in the exercise group, indicating that M2 macrophages and angiogenesis are related to accelerated wound healing. These findings suggest that moderate-intensity exercise increases TGF-ß1 derived from M2 macrophages, which may be associated with enhanced angiogenesis and wound healing in young mice.

Tumor suppression and improvement in immune systems by specific activation of dopamine D1-receptor-expressing neurons in the nucleus accumbens.

Recent research has suggested that the mesolimbic dopamine network that mainly terminates in the nucleus accumbens may positively control the peripheral immune system. The activation of dopamine receptors in neurons in the nucleus accumbens by the release of endogenous dopamine is thus expected to contribute to efferent immune regulation. As in the stimulation of Gs-coupled dopamine D1-receptors or Gi-coupled D2-receptors by endogenous dopamine, we investigated whether specific stimulation of dopamine D1-receptor-expressing neurons or inhibition of dopamine D2-receptor-expressing neurons in the nucleus accumbens could produce anti-tumor effects and improve the immune system in transgenic mice using pharmacogenetic techniques. Repeated stimulation of D1-receptor-expressing neurons in either the medial shell, lateral shell or core regions of the nucleus accumbens significantly decreased tumor volume under a state of tumor transplantation, whereas repeated suppression of D2-receptor-expressing neurons in these areas had no effect on this event. The number of splenic CD8+ T cells was significantly increased following repeated stimulation of D1-receptor-expressing neurons in the nucleus accumbens of mice with tumor transplantation. Furthermore, this stimulation produced a significant reduction in the population of splenic CD8+ T cells that expressed immune checkpoint-related inhibitory receptors, PD-1, TIM-3 and LAG-3. These findings suggest that repeated stimulation of D1-receptor-expressing neurons (probably D1-receptor-expressing medium spiny neurons) in the nucleus accumbens suppressed tumor progression and improved the immune system by suppressing the exhaustion of splenic CD8+ T cells.

Plastic changes in amygdala subregions by voluntary running contribute to exercise-induced hypoalgesia in neuropathic pain model mice.

Physical exercise has been established as a low-cost, safe, and effective way to manage chronic pain, but exact mechanisms underlying such exercise-induced hypoalgesia (EIH) are not fully understood. Since a growing body of evidence implicated the amygdala (Amyg) as a critical node in emotional affective aspects of chronic pain, we hypothesized that the Amyg may play important roles to produce EIH effects. Here, using partial sciatic nerve ligation (PSL) model mice, we investigated the effects of voluntary running (VR) on the basal amygdala (BA) and the central nuclei of amygdala (CeA). The present study indicated that VR significantly improved heat hyperalgesia which was exacerbated in PSL-Sedentary mice, and that a significant positive correlation was detected between total running distances after PSL-surgery and thermal withdrawal latency. The number of activated glutamate (Glu) neurons in the medal BA (medBA) was significantly increased in PSL-Runner mice, while those were increased in the lateral BA in sedentary mice. Furthermore, in all subdivisions of the CeA, the number of activated gamma-aminobutyric acid (GABA) neurons was dramatically increased in PSL-Sedentary mice, but these numbers were significantly decreased in PSL-Runner mice. In addition, a tracer experiment demonstrated a marked increase in activated Glu neurons in the medBA projecting into the nucleus accumbens lateral shell in runner mice. Thus, our results suggest that VR may not only produce suppression of the negative emotion such as fear and anxiety closely related with pain chronification, but also promote pleasant emotion and hypoalgesia. Therefore, we conclude that EIH effects may be produced, at least in part, via such plastic changes in the Amyg.

Bilateral increases in ERK activation at the spinomedullary junction region by acute masseter muscle injury during temporomandibular joint inflammation in the rats.

We determined the role of persistent monoarthritis of temporomandibular joint region (TMJ) on bilateral masseter muscle (MM) nociception in male rats using orofacial nocifensive behaviors, phosphorylated extracellular signal-regulated kinase and Fos induction at the trigeminal subnucleus caudalis/upper cervical spinal cord (Vc/C2) region in response to formalin injection to the MM region. TMJ inflammation was induced by local injection of CFA into the left TMJ region. Orofacial nocifensive behaviors evoked by formalin injection ipsilateral or contralateral to the TMJ inflammation appeared to be increased at 1-14 days or at 1, 10 and 14 days after induction of TMJ inflammation, respectively, while increases in behavioral duration were seen mainly in the late phase rather than the early phase. The number of pERK positive cells was investigated in superficial laminae at the Vc/C2 region at 3, 10, 20, 60 and 80 min after MM stimulation with formalin at 14 days after TMJ inflammation. TMJ-inflamed rats displayed greater responses of pERK expression by the ipsilateral MM stimulation at 3-60 min, while contralateral MM stimulation increased pERK expression at 3, 10 and 20 min compared to non-CFA rats. Fos expression by MM stimulation was increased at 14 days after induction of TMJ inflammation regardless of the affected side. These findings showed that persistent TMJ inflammation for 10 and 14 days is sufficient to enhance MM nociception indicated by behaviors and neural responses in superficial laminae at the Vc/C2 region.

A new aspect of chronic pain as a lifestyle-related disease.

Physical exercise has been established as a low-cost, safe, and effective way to manage chronic intractable pain. We investigated the underlying mechanisms of exercise-induced hypoalgesia (EIH) using a mouse model of neuropathic pain (NPP). Epigenetic changes in activated microglia and maintained GABA synthesis in the spinal dorsal horn may contribute to EIH. Voluntary exercise (VE), a strong reward for animals, also induced EIH, which may be due in part to the activation of dopamine (DA) neurons in the ventral tegmental area (VTA). VE increases the expression of pCREB in dopaminergic neurons in the VTA, which would enhance dopamine production, and thereby contributes to the activation of the mesolimbic reward system in NPP model mice. We demonstrated that neurons in the laterodorsal tegmental and pedunculopontine tegmental nuclei, a major input source of rewarding stimuli to the VTA, were activated by exercise. Chronic pain is at least partly attributed to sedentary and inactive lifestyle as indicated by the Fear-avoidance model. Therefore, chronic pain could be recognized as a lifestyle-related disease. Physical activity/inactivity may be determined by genetic/epigenetic and neural factors encoded in our brain. The hypothalamus and reward system is closely related in the axis of food intake, energy metabolism and physical activity. Understanding the interactions between the mesolimbic DA system and the hypothalamus that sense and regulate energy balance is thus of significant importance. For example, proopiomelanocortin neurons and melanocortin 4 receptors may play a role in connecting these two systems. Therefore, in a certain sense, chronic pain and obesity may share common behavioral and neural pathology, i.e. physical inactivity, as a result of inactivation of the mesolimbic DA system. Exercise and increasing physical activity in daily life may be important in treating and preventing chronic pain, a life-style related disease.

Histone Acetylation in Microglia Contributes to Exercise-Induced Hypoalgesia in Neuropathic Pain Model Mice.

UNLABELLED: Physical exercise can attenuate neuropathic pain (NPP), but the exact mechanism underlying exercise-induced hypoalgesia (EIH) remains unclear. Recent studies have shown that histone hyperacetylation via pharmacological inhibition of histone deacetylases in the spinal cord attenuates NPP, and that histone acetylation may lead to the production of analgesic factors including interleukin 10. We intended to clarify whether histone acetylation in microglia in the spinal dorsal horn contributes to EIH in NPP model mice. C57BL/6J mice underwent partial sciatic nerve ligation (PSL) and PSL- and sham-runner mice ran on a treadmill at a speed of 7 m/min for 60 min/d, 5 days per week, from 2 days after the surgery. PSL-sedentary mice developed mechanical allodynia and heat hyperalgesia, but such behaviors were significantly attenuated in PSL-runner mice. In immunofluorescence analysis, PSL surgery markedly increased the number of histone deacetylase 1-positive/CD11b-positive microglia in the ipsilateral superficial dorsal horn, and they were significantly decreased by treadmill-running. Moreover, the number of microglia with nuclear expression of acetylated H3K9 in the ipsilateral superficial dorsal horn was maintained at low levels in PSL-sedentary mice, but running exercise significantly increased them. Therefore, we conclude that the epigenetic modification that causes hyperacetylation of H3K9 in activated microglia may play a role in producing EIH. PERSPECTIVE: This article presents the importance of epigenetic modification in microglia in producing EIH. The current research is not only helpful for developing novel nonpharmacological therapy for NPP, but will also enhance our understanding of the mechanisms and availability of exercise in our daily life.

Lack of oncostatin M receptor ß leads to adipose tissue inflammation and insulin resistance by switching macrophage phenotype.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, plays important roles in a variety of biological functions, including inflammatory responses. However, the roles of OSM in metabolic diseases are unknown. We herein analyzed the metabolic parameters of OSM receptor ß subunit-deficient (OSMRß(-/-)) mice under normal diet conditions. At 32 weeks of age, OSMRß(-/-) mice exhibited mature-onset obesity, severer hepatic steatosis, and insulin resistance. Surprisingly, insulin resistance without obesity was observed in OSMRß(-/-) mice at 16 weeks of age, suggesting that insulin resistance precedes obesity in OSMRß(-/-) mice. Both OSM and OSMRß were expressed strongly in the adipose tissue and little in some other metabolic organs, including the liver and skeletal muscle. In addition, OSMRß is mainly expressed in the adipose tissue macrophages (ATMs) but not in adipocytes. In OSMRß(-/-) mice, the ATMs were polarized to M1 phenotypes with the augmentation of adipose tissue inflammation. Treatment of OSMRß(-/-) mice with an anti-inflammatory agent, sodium salicylate, improved insulin resistance. In addition, the stimulation of a macrophage cell line, RAW264.7, and peritoneal exudate macrophages with OSM resulted in the increased expression of M2 markers, IL-10, arginase-1, and CD206. Furthermore, treatment of C57BL/6J mice with OSM increased insulin sensitivity and polarized the phenotypes of ATMs to M2. Thus, OSM suppresses the development of insulin resistance at least in part through the polarization of the macrophage phenotypes to M2, and OSMRß(-/-) mice provide a unique mouse model of metabolic diseases.

Regulation of AMP-activated protein kinase signaling by AFF4 protein, member of AF4 (ALL1-fused gene from chromosome 4) family of transcription factors, in hypothalamic neurons.

In the hypothalamus, fasting induces a member of the AF4 family of transcription factors, AFF4, which was originally identified as a fusion partner of the mixed-lineage leukemia gene in infant acute lymphoblastic leukemia. However, the roles of AFF4 in the hypothalamus remain unclear. We show herein that expression of AFF4 increased upon addition of ghrelin and fasting in the growth hormone secretagogue receptor-expressing neurons of the hypothalamus. In the growth hormone secretagogue receptor-expressing hypothalamic neuronal cell line GT1-7, ghrelin markedly induced expression of AFF4 in a time- and dose-dependent manner. Overexpression of AFF4 in GT1-7 cells specifically induced expression of the AMP-activated protein kinase (AMPK) α2 subunit but failed to induce other AMPK subunits and AMPK upstream kinases. The promoter activity of the AMPKα2 gene increased upon addition of AFF4, suggesting that AFF4 regulates transcription of the AMPKα2 gene. Additionally, AFF4 also increased the phosphorylation of acetyl-CoA carboxylase α (ACCα), a downstream target of AMPK. In GT1-7 cells, ghrelin phosphorylated ACCα through AMPKα phosphorylation in the early phase (15 min) of the activation. However, ghrelin-induced expression of AMPKα2 and phosphorylation of ACCα in the late phase (2 h) of the activation were independent of AMPKα phosphorylation. Attenuation of expression of AFF4 by its siRNA in GT1-7 cells decreased ghrelin-induced AMPKα2 expression and ACCα phosphorylation in the late phase of the activation. AFF4 may therefore help to maintain activation of AMPK downstream signaling under conditions of prolonged stimulation with ghrelin, such as during fasting.

Site-specific subtypes of macrophages recruited after peripheral nerve injury.

After partial ligation of mouse sciatic nerve, the subtypes of macrophages were examined in the injured nerve and dorsal root ganglia (DRGs). Many M1 macrophages, which were inducible nitric oxide synthase (iNOS)-positive and arginase-1 (Arg-1)-negative, and neutrophils infiltrated the injured nerve. In contrast, almost all macrophages infiltrating the ipsilateral side of DRGs after the nerve injury were iNOS⁻/Arg-1⁺, M2 type. The infiltration of M1 and M2 macrophages was first observed in the injured nerve and ipsilateral DRGs on days 1 and 2, respectively. In addition, the macrophage infiltration preceded the activation of microglia in the ipsilateral dorsal horn of spinal cord. Thus, infiltrating macrophages after peripheral nerve injury may play unique roles dependent on the location in the development of neuropathic pain.

Deprivation of anticipated food under scheduled feeding induces c-Fos expression in the caudal part of the arcuate nucleus of hypothalamus through histamine H1 receptors in rats: potential involvement of E3 subgroup of histaminergic neurons in tuberomammillary nucleus.

It is well established that histaminergic neurons densely innervate the anterior hypothalamus and regulate several functions through histamine H(1) receptor (H1R). However, functional innervations of histaminergic neurons in the caudal hypothalamus have been poorly investigated. Recently, we have demonstrated that c-Fos, a marker of neuronal activation, was significantly induced by food deprivation under scheduled feeding in H1R-expressing cells in the caudal part of the arcuate nucleus of hypothalamus (cARC) of rats and histaminergic neurons innervating this area. In this study, we have examined the functional involvement of histaminergic neurons in the food deprivation-induced c-Fos expression in the cARC under scheduled feeding. The c-Fos expression in the cARC by food deprivation was significantly suppressed by pretreatment with antihistamines. After food deprivation, the number of c-Fos-histidine decarboxylase (HDC) double-positive neurons was mostly increased in the E3 subdivision of the tuberomammillary nucleus (TM). Under the restricted feeding schedule, significant expressions of c-Fos were detected in the TM and cARC only when rats strongly anticipated feeding, compared with a slight c-Fos induction in both nuclei when they were satiated. These findings suggest that the histaminergic neurons in the E3 subdivision of the TM are selectively activated by deprivation of an anticipated food under scheduled feeding and functionally innervate the H1R-expressing neurons in the cARC.

[Limbic system and autonomic nervous system].

The viral transneuronal labeling method using pseudorabies virus (PRV) is an ideal technique for identifying the central sites that regulate the sympathetic nervous system. Regions were identified in limbic system such as extended amygdaloid complex, lateral septum, infralimbic, insular, ventromedial temporal cortical regions, as well as in several hypothalamic and brain stem nuclei. Emotional stress causes rapid and transient expression of immediate early genes (IEGs) such as c-Fos in the brain, and the monitoring of IEGs has enabled the visualization of the neurocircuitry of stress. By a comparison of the data from the separate PRV and c-Fos neuroanatomical labeling techniques, the central sites which regulate emotional stress-induced sympathoadrenal activation can be deduced. Estrogen receptors are expressed in the brain, where estrogen modulates central nervous function and autonomic nervous function. Estrogen attenuated the stress-induced c-Fos expression in medial amygdaloid nucleus, paraventricular hypothalamic nucleus; these same regions contain central sympathetic neurons and neurons with immunoreactive estrogen receptors.

TROY, a novel member of the tumor necrosis factor receptor superfamily in the central nervous system.

Using a signal sequence trap method, we isolated TROY, a novel member of the tumor necrosis factor receptor superfamily (TNFRSF), from a mouse brain cDNA library. TROY mRNA is strongly expressed in brain and embryo. In situ hybridization analysis of the embryo showed that TROY mRNA was exclusively expressed in the epithelium of many tissues, including neuroepithelium. In the developing central nervous system, TROY mRNA was strongly expressed in the ventricular and subventricular zones, which contain neuronal and glial precursors during mouse embryogenesis that are both region-specific and stagedependent. In addition, TROY mRNA was expressed in the developing olfactory bulb from embryonic day (E) 13.5 to neonate. Next, we focused on the detailed cellular characterization of TROY-expressing cells in the developing olfactory system.TROYmRNAwas first detected in the olfactory nerve layer (ONL) of the olfactory bulb at E13.5 and was expressed most intensely in the inner ONL (ONL-i) during late embryogenesis. In the postnatal olfactory bulb, TROY-expressing cells were also detected in the glomerular layer (GL) and ONL-i. TROY was intensely expressed in olfactory ensheathing cells (OECs) of the ONL-i, which are positive for neuropeptide Y (NPY), but negative for S-100 or p75 low-affinity nerve growth factor receptor. Furthermore, TROY was also detected in glial fibrillary acidic protein (GFAP)-positive glial cells of the ONL-i and GL. Thus, TROY was expressed in some specific subsets of glial cells in the olfactory bulb, including OECs, and may play some roles in the developing and adult olfactory system.

[Descending facilitation in chronic stress and chronic pain state].

The spino-thalamic tract consists of two systems; the lateral system terminates in the somato-sensory cortex, and participates in the sensory discrimination of pain, and the medial system terminates in the anterior cingulated cortex (ACC) and insular cortex (IC) to mediate affective components of pain. Persistent pain induces plastic changes in cortical neurons, especially in the ACC and IC. Activation of these neurons is transmitted to the periaqueductal gray and rostroventromedial medulla (RVM) (descending pain control system). This system has long been considered to exert descending inhibition, but recent studies revealed that it also causes facilitation in certain pathological conditions. A variety of stressful stimuli have been shown to affect pain sensitivity. We demonstrated that chronic restraint stress induced thermal hyperalgesia in rats, in which phosphorylated ERK and levels of tryptophan hydroxylase, a key enzyme of 5-HT production, were increased in the RVM. 5HT released from the bulbospinal neurons may exert facilitatory effects on spinal nociceptive processing probably through 5HT3 receptors. Patients suffering chronic pain originating from deep tissues, such as temporo-mandibular disorder, fibromyalgia, or low back pain, often complain of pain and tenderness in various parts of the body. We injected complete Freund's adjuvant into a temporo-mandibular joint of rats unilaterally, and then injected 5% formalin into the ipsilateral or contralateral masseter muscle 2 weeks later. Pain-related behavior and neuronal activation in the spinal trigeminal nucleus were enhanced on both sides compared to those in non-inflammatory controls. Systemic enhancement of pain and hyperalgesia induced by unilateral joint inflammation may have been caused by the central sensitization and descending facilitation.

Perturbed intraepithelial differentiation of corneal epithelium in c-Fos-null mice.

PURPOSE: AP-1 is a transcription factor that plays a pivotal role in regulating cellular homeostasis and which may modulate the differentiation of corneal epithelial cells. We examined the role of c-Fos in the differentiation of corneal epithelial cells by using c-Fos-deficient (c-fos (-/-)) mice. METHODS: Ten adult c-fos (-/-) mice and ten control (c-fos (+/-) or c-fos (+/+)) mice were used. The expression patterns of the mRNA and protein of keratin 12 (K12) were determined to examine the differentiation of cornea-type epithelium. To evaluate the intraepithelial differentiation from basal cells to superficial cells, the ultrastructure of the corneal epithelium was studied. We focused on the formation of desmosomes in the superficial, suprabasal, and basal cell layers, and also on the hemidesmosomes. The number of desmosomes in each epithelial layer was statistically analyzed by using an unpaired t test. The expressions of keratin 14 (K14), desmoglein, E-cadherin, occludin, connexin 43, filaggrin, loricrin, and involucrin were examined to analyze epithelial differentiation. RESULTS: The mRNA and protein of K12 were expressed in the corneal epithelium of c-fos (-/-) and control mice. Ultrastructural observations showed that the number of desmosomes between the basal cells of the corneal epithelia was similar in c-fos (-/-) and control mice. However, there were fewer desmosomes between suprabasal cells and between superficial cells in c-fos (-/-) mice than in control mice. The number of hemidesmosomes in the corneal epithelial cells in c-Fos-null mice was similar to that in control mice. The expressions of the other epithelial cell differentiation markers were not affected by the absence of c-Fos. Ultrastructural observations showed a disarrangement of the corneal epithelium in the c-Fos-null mice. CONCLUSIONS: The absence of c-Fos disturbs the formation of desmosomes in the superficial layers of the corneal epithelium, suggesting a perturbation of intraepithelial differentiation from the basal epithelial cells to the suprabasal and superficial epithelial cells.

AP-1 expression in ethanol-treated corneal epithelium in vivo.

PURPOSE: To examine the expression pattern of stress-related genes, c-fos and c-jun, both the major components of activator protein-1 (AP-1), in rat corneal epithelium treated with a short-term ethanol exposure. The purpose of the current study was to examine if the ethanol exposure during laser epithelial keratomileusis (LASEK) may stimulate or damage the corneal epithelial cells. METHOD: Sixty male Wistar rats were used. Fifty microliters of 20% ethanol was placed onto a surface 2.4 mm in diameter of the central corneal epithelium for 30 s. The affected eyes, washed with saline, were then enucleated after various intervals of healing. To know the expression pattern of c-fos and c-jun mRNAs and c-Fos, c-Jun and Jun D proteins, in situ hybridization and immunohistochemistry were carried out. The expression level of c-fos and c-jun mRNAs was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Apoptotic nuclei in the tissue sections were identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. RESULTS: Thirty to 60 min after the treatment, c-fos and c-jun mRNAs were detected in the corneal epithelium. These signals were no longer evident at 90 min. c-Fos protein was detected in the corneal epithelium around the area of ethanol exposure from 60 to 120 min after the treatment, while c-Jun protein was not detected. Jun D protein was detected in control whole corneal epithelium and not affected by ethanol exposure in the periphery. The levels of c-fos and c-jun mRNAs were increased approximately 8 times at 30 min compared with the control level. TUNEL-positive apoptotic nuclei in the tissue sections were identified. CONCLUSION: Corneal epithelial cells, especially those surrounding the ethanol-exposed area, are transiently transcriptionally activated at a very early phase after the ethanol exposure. mRNA expression for c-fos is followed by protein synthesis, but that of c-jun is not followed by protein synthesis. Resistance of Jun D protein expression to ethanol suggests that it might be a candidate for an AP-1 complex with c-Fos.

Changes in estrogen receptors alpha and beta expression in the brain of mice exposed prenatally to bisphenol A.

The expression of ERs alpha and beta and serotonergic neurons were evaluated in the brains of mice prenatally exposed to Bisphenol A, a known endocrine disrupting chemical (EDc). Bisphenol A was administered orally at a dose of 2ng/g body weight on gestinational days 11-17 to pregnant ICR mice. Newborn male offspring (Bis-A mice) were evaluated for the immunoreactivity of ERs alpha and beta, serotonin, and serotonin transporter positive cells in the dorsal raphe nucleus (DRN). The serum testosterone level was also evaluated. In the Bis-A mice, the expression of ERs alpha and beta at 5 and 13 weeks was increased compared with the controls (P<0.04), but this difference disappeared by the 9th week. The serotonin, serotonin transporter, and testosterone level differences between two groups did not reach significance. Exposure to bisphenol A may have changed the expression of ERs in the brain, but did not directly affect serotonin neurons in the DRN.

Characterization of TROY-expressing cells in the developing and postnatal CNS: the possible role in neuronal and glial cell development.

A member of the tumor necrosis factor receptor superfamily, TROY, is expressed in the CNS of embryonic and adult mice. In the present study, we characterized TROY-expressing cells in the embryonic and postnatal forebrain. In the early embryonic forebrain, TROY was highly expressed in nestin-positive neuroepithelial cells and radial glial cells, but not in microtubule-associated protein 2-positive postmitotic neurons. During the late embryonic and postnatal development, expression of TROY was observed in radial glial cells and astrocytes, whereas its expression was not detected in neuronal lineage cells. In addition, TROY was exclusively expressed in Musashi-1-positive multipotent/glial progenitors in the postnatal subventricular zone. To investigate the functions of TROY in neural development, we overexpressed TROY in PC12 cells and established stably expressing cell clones. As expected, the signals from overexpressed TROY were constitutively transduced via the activation of the nuclear factor-kappaB and the c-Jun N-terminal kinase pathways in such clones. In addition, upregulation of negative basic helix-loop-helix transcription factors, HES-5 and Id2 proteins, was observed in the TROY-overexpressing clones. Interestingly, the overexpression of TROY in PC12 cells strongly inhibited nerve growth factor-induced neurite outgrowth with reduction of some markers of differentiated neurons, such as neurofilament 150 kDa and neuron-specific beta-tubulin. These findings suggest that the signaling from TROY regulates neuronal differentiation at least in part.

Characterization of TROY/TNFRSF19/TAJ-expressing cells in the adult mouse forebrain.

A member of the tumor necrosis factor receptor superfamily (TNFRSF), TROY/TNFRSF19/TAJ, is highly expressed in the brain of adult mice. Northern blot analysis using mRNA taken from regions of the adult CNS showed the expression of TROY in all regions examined, including the olfactory bulb, cerebral cortex, striatum, and hippocampus. In situ hybridization and immunohistochemistry revealed that TROY mRNA and protein were strongly expressed in the rostral migratory stream (RMS) and subventricular zone (SVZ) of adult mice. In the adult SVZ, some glial fibrillary acidic protein (GFAP)-positive cells (type B cells) are thought to be multipotent neural stem cells. These type B cells divide slowly and generate epidermal growth factor receptor (EGFR)-positive transit-amplifying precursor cells (type C cells) in the presence of epidermal growth factor (EGF). Type C cells give rise to neuron-specific class III beta-tubulin (TuJ1)-positive neuroblasts (type A cells) that migrate to the olfactory bulb along the RMS. TROY-expressing cells were GFAP-positive, EGFR-positive, and TuJ1-negative in the adult SVZ. From these findings, TROY appears to be expressed in type B and type C cells, but not in type A cells, which was supported by immunoelectron microscopy. In addition, TROY was expressed in GFAP-positive astrocytes of the various regions, such as the cerebral cortex, striatum, and hippocampus. Thus, TROY was expressed in uncommitted precursor cells and astroglial lineage cells, suggesting that TROY plays some roles in the regulation of gliogenesis in the adult CNS.

Acute suppression, but not chronic genetic deficiency, of c-fos gene expression impairs long-term memory in aversive taste learning.

Several lines of evidence have indicated that the establishment of long-term memory requires protein synthesis, including the synthesis of immediate-early gene products. Although the anatomical expression patterns of the c-fos gene, a transcription factor-encoding immediate-early gene, in conditioned taste aversion (CTA) are well documented, the functional roles of c-fos gene expression and Fos-mediated transcription remain to be clarified. Using the antisense oligodeoxynucleotide (AS-ODN) method in rats and gene-targeting knockout techniques in mice (c-fos(-/-) mice), we examined the roles of c-fos gene expression in the acquisition, retrieval, and retention of CTA. Preconditioning microinfusion of AS-ODN directed against c-fos mRNA (c-fos AS-ODN) into the parabrachial nucleus (PBN) impaired the acquisition, whereas infusion of control ODNs consisting of a randomized or inverted base order had no effect. Microinfusion of c-fos AS-ODN into either the amygdala or insular cortex did not impair the acquisition, whereas it attenuated the retention. Retrieval and subsequent retention of an acquired CTA were not disrupted by c-fos AS-ODN infusion into the PBN or amygdala. Microinfusion of another AS-ODN directed against zif268 (egr-1, krox-24, NGFI-A) mRNA into the PBN or amygdala did not affect the acquisition and retention. The genetic deficiency in c-fos(-/-) mice caused normal acquisition and retention. The present results suggest that the Fos-mediated gene transcription in the PBN, amygdala, or insular cortex plays critical roles in the acquisition and/or consolidation, but not the retrieval, of long-term taste memory; nevertheless, some other factors could compensate CTA mechanism when Fos-mediated transcription is not available.