Effects of electroacupuncture on stress-induced gastric dysrhythmia and mechanisms involving autonomic and central nervous systems in functional dyspepsia.

Electroacupuncture (EA) is widely used as an effective method to treat stress-related disorders. However, its mechanisms remain largely unknown. The aim of this study was to investigate the effects and mechanisms of EA on gastric slow wave (GSW) dysrhythmia and c-Fos expression in the nucleus of the solitary tract (NTS) induced by stress in a rodent model of functional dyspepsia (FD). Rats in the neonatal stage were treated using intragastric iodoacetamide. Eight weeks later, the rats were implanted with electrodes in the stomach for the measurement of GSW and electrodes into accupoints ST36 for EA. Autonomic functions were assessed by spectral analysis of heart rate variability. Rats were placed for 30 min in a cylindrical plastic tube for acute restraint stress. The involvement of a central afferent pathway was assessed by measuring c-Fos-immunoreactive cells in the NTS. 1) EA normalized restraint stress-induced impairment of GSW in FD rats. 2) EA significantly increased vagal activity (P = 0.002) and improved sympathovagal balance (P = 0.004) under stress in FD rats. 3) In FD rats under restraint stress, plasma norepinephrine concentration was increased substantially (P < 0.01), which was suppressed with EA. 4) The EA group showed increased c-Fos-positive cell counts in the NTS compared with the sham EA group (P < 0.05) in FD rats. Acute restraint stress induces gastric dysrhythmia in a rodent model of FD. EA at ST36 improves GSW under stress in FD rats mediated via the central and autonomic pathways, involving the NTS and vagal efferent pathway.

Effects of histone acetyltransferase inhibitors on L-DOPA-induced dyskinesia in a murine model of Parkinson's disease.

Histone acetylation is a key regulatory factor for gene expression in cells. Modulation of histone acetylation by targeting of histone acetyltransferases (HATs) effectively alters many gene expression profiles and synaptic plasticity in the brain. However, the role of HATs on L-DOPA-induced dyskinesia of Parkinson's disease (PD) has not been reported. Our aim was to determine whether HAT inhibitors such as anacardic acid, garcinol, and curcumin from natural plants reduce severity of L-DOPA-induced dyskinesia using a unilaterally 6-hydroxydopamine (6-OHDA)-lesioned PD mouse model. Anacardic acid 2 mg/kg, garcinol 5 mg/kg, or curcumin 100 mg/kg co-treatment with L-DOPA significantly reduced the axial, limb, and orofacial (ALO) score indicating less dyskinesia with administration of HAT inhibitors in 6-OHDA-lesioned mice. Additionally, L-DOPA's efficacy was not altered by the compounds in the early stage of treatment. The expression levels of c-Fos, Fra-2, and Arc were effectively decreased by administration of HAT inhibitors in the ipsilateral striatum. Our findings indicate that HAT inhibitor co-treatment with L-DOPA may have therapeutic potential for management of L-DOPA-induced dyskinesia in patients with PD.

Topical dura mater application of CFA induces enhanced expression of c-fos and glutamate in rat trigeminal nucleus caudalis: attenuated by KYNA derivate (SZR72).

BACKGROUND: Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished by a systemic administration of kynurenic acid (KYNA) derivate (SZR72). Here, we hypothesize that this activation may extend to the trigeminal complex in the brainstem and is attenuated by treatment with SZR72. METHODS: Activation in the trigeminal nucleus caudalis (TNC) and the trigeminal tract (Sp5) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1ß and TNFα in the TNC/Sp5 and other regions of the brainstem and at the C1-C2 regions of the spinal cord. RESULTS: We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C1-C2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6 and IL-1ß immunopositivity were detected in fibers of Sp5 and neither of these molecules showed any change in immunoreactivity following CFA administration. CONCLUSION: This is the first study demonstrating that dural application of CFA increases the expression of c-fos and glutamate in TNC neurons. Treatment with the KYNA analogue prevented this expression.

Inhibition of RANKL-induced osteoclast differentiation through the downregulation of c-Fos and NFATc1 by Eremochloa ophiuroides (centipedegrass) extract.

Osteoclasts, derived from hematopoietic stem cells, are specialized macrophages and have a homeostatic role in skeletal modeling and remodeling with bone-forming osteoblasts. However, excessive osteoclast activity induces bone diseases, including osteoporosis, periodontitis and rheumatoid arthritis. Natural substances have received attention as therapeutic drugs in human diseases. In the current study, cells isolated from mouse bone marrow, and a mouse model, were used to determine the effect of centipedegrass extract (CGE) on osteoclasts. Multiple concentrations of CGE were administered to bone marrow cells for 24­72 hours and, for the in vivo study, mice were treated with CGE for 8 days. The effects of CGE on transcription and translation of osteoclast-associated molecules were then determined using reverse transcription-polymerase chain reaction and immunoblotting, respectively. In the present study it was shown that CGE extracted from Eremochloa ophiuroides (centipedegrass) inhibited receptor activator of nuclear factor κ­B ligand (RANKL)­mediated osteoclast differentiation in bone marrow macrophages, without cytotoxicity, in a dose­dependent manner. CGE decreased the expression levels of osteoclast­specific genes, including matrix metalloproteinase­9, osteoclast­associated immunoglobulin­like receptor and cathepsin K, however, CGE had no inhibitory effect on the expression levels of mitogen­activated protein kinases, nuclear factor­κB and Akt. Furthermore, the protein and RNA levels of RANKL­induced c­Fos and nuclear factor of activated T-cell cytoplasmic 1 were suppressed by CGE. These results indicated that CGE may serve as a useful drug in the prevention of bone loss.

Suckling-induced Fos activation and melanin-concentrating hormone immunoreactivity during late lactation.

AIMS: Melanin-concentrating hormone (MCH) is implicated in the control of food intake, body weight regulation and energy homeostasis. Lactation is an important physiological model to study the hypothalamic integration of peripheral sensory signals, such as suckling stimuli and those related to energy balance. MCH can be detected in the medial preoptic area (MPOA), especially around the 19th day of lactation, when this hormone is described as displaying a peak synthesis followed by a decrease after weaning. The physiological significance of this phenomenon is unclear. Therefore, we aimed to investigate hypothalamic changes associated to sensory stimulation by the litter, in special its influence over MCH synthesis. MAIN METHODS: Female Wistar rats (n=56) were euthanized everyday from lactation days 15-21, with or without suckling stimulus (WS and NS groups, respectively). MCH and Fos immunoreactivity were evaluated in the MPOA and lateral and incerto-hypothalamic areas (LHA and IHy). KEY FINDINGS: Suckling stimulus induced Fos synthesis in all regions studied. An increase on the number of suckling-induced Fos-ir neurons could be detected in the LHA after the 18th day. Conversely, the amount of MCH decreased in the MPOA from days 15-21, independent of suckling stimulation. No colocalization between MCH and Fos could be detected in any region analyzed. SIGNIFICANCE: Suckling stimulus is capable of stimulating hypothalamic regions not linked to maternal behavior, possibly to mediate energy balance aspects of lactation. Although dams are hyperphagic before weaning, this behavioral change does not appear to be mediated by MCH.

Circadian phenotyping of obese and diabetic db/db mice.

Growing evidence links metabolic disorders to circadian alterations. Genetically obese db/db mice, lacking the long isoform of leptin receptor, are a recognized model of type 2 diabetes. In this study, we aimed at characterizing the potential circadian alterations of db/db mice in comparison to db/+ control mice. By using telemetry devices, we first reported arrhythmicity in general activity of most db/db mice under both light-dark cycle and constant darkness, while their rhythm of body temperature is less dramatically disrupted. Water access restricted to nighttime restores significant rhythmicity in behaviorally arrhythmic db/db mice, indicating a masking effect of polydipsia when water is available ad libitum. Endogenous period of temperature rhythm under constant dark conditions is significantly increased (+30 min) in db/db compared with db/+ mice. Next, we studied the oscillations of clock proteins (PER1, PER2 and BMAL1) in the suprachiasmatic nuclei (SCN), the site of the master clock, and detected no difference according to the genotype. Furthermore, c-FOS and P-ERK1/2 expression in response to a light pulse in late night was significantly increased (+80 and +55%, respectively) in the SCN of these diabetic mice. We previously showed that, in addition to altered activity rhythms, db/db mice exhibit altered feeding rhythm. Therefore, we investigated daily patterns of clock protein expression in medial hypothalamic oscillators involved in feeding behavior (arcuate nucleus, ventro- and dorso-medial hypothalamic nuclei). Compared with db/+ mice, very subtle or no difference in oscillations of PER1 and BMAL1 is found in the medial hypothalamus. Although we did not find a clear link between altered hypothalamic clockwork and behavioral rhythms in db/db mice, our results highlight a lengthened endogenous period and altered photic integration in these genetically obese and diabetic mice.

Chlorella vulgaris reduces the impact of stress on hypothalamic-pituitary-adrenal axis and brain c-fos expression.

Predominantly emotional stressors activate a wide range of brain areas, as revealed by the expression of immediate early genes, such as c-fos. Chlorella vulgaris (CV) is considered a biological response modifier, as demonstrated by its protective activities against infections, tumors and stress. We evaluated the effect of acute pretreatment with CV on the peripheral and central responses to forced swimming stress in adult male rats. Pretreatment with CV produced a significant reduction of stress-related hypothalamic-pituitary-adrenal activation, demonstrated by decreased corticotrophin releasing factor gene expression in the hypothalamic paraventricular nucleus (PVN) and lower ACTH response. Hyperglycemia induced by the stressor was similarly reduced. This attenuated neuroendocrine response to stress occurred in parallel with a diminished c-fos expression in most evaluated areas, including the PVN. The data presented in this study reinforce the usefulness of CV to diminish the impact of stressors, by reducing the HPA response. Although our results suggest a central effect of CV, further studies are necessary to understand the precise mechanisms underpinning this effect.

Central apelin mediates stress-induced gastrointestinal motor dysfunction in rats.

Apelin, an endogenous ligand for APJ receptor, has been reported to be upregulated in paraventricular nucleus (PVN) following stress. Central apelin is known to stimulate release of corticotropin-releasing factor (CRF) via APJ receptor. We tested the hypothesis that stress-induced gastrointestinal (GI) dysfunction is mediated by central apelin. We also assessed the effect of exogenous apelin on GI motility under nonstressed (NS) conditions in conscious rats. Prior to solid gastric emptying (GE) and colon transit (CT) measurements, APJ receptor antagonist F13A was centrally administered under NS conditions and following acute stress (AS), chronic homotypic stress (CHS), and chronic heterotypic stress (CHeS). Plasma corticosterone was assayed. Strain gage transducers were implanted on serosal surfaces of antrum and distal colon to record postprandial motility. Stress exposure induced coexpression of c-Fos and apelin in hypothalamic PVN. Enhanced hypothalamic apelin and CRF levels in microdialysates were detected following AS and CHeS, which were negatively and positively correlated with GE and CT, respectively. Central F13A administration abolished delayed GE and accelerated CT induced by AS and CHeS. Central apelin-13 administration increased the plasma corticosterone and inhibited GE and CT by attenuating antral and colonic contractions. The inhibitory effect elicited by apelin-13 was abolished by central pretreatment of CRF antagonist CRF9-41 in antrum, but not in distal colon. Central endogenous apelin mediates stress-induced changes in gastric and colonic motor functions through APJ receptor. The inhibitory effects of central exogenous apelin-13 on GI motility appear to be partly CRF dependent. Apelin-13 inhibits colon motor functions through a CRF-independent pathway.

Fourth ventricular CART peptide induces c-fos in the area postrema and nucleus of the solitary tract via a CRF-receptor dependent mechanism.

Cocaine-and amphetamine-regulated transcript peptides (CARTp) suppress gastric emptying and nutritional intake following 4th icv administration. Whereas, the CARTp inhibition of gastric emptying was blocked by pre-treatment with a non-selective corticotropin releasing factor (CRF) antagonist, sucrose drinking was not, suggesting that CARTp- and CRF controls for food intake and gastric emptying are operated through separable dorsal hindbrain mechanisms. The aim of the study was to explore CARTp-CRF brainstem interactions on patterns of neuronal activation in areas of the brainstem and midbrain relevant to gastrointestinal control and feeding regulation. Rats received 4th icv injections of combinations of vehicle, CARTp (1µg), or the nonselective CRF antagonist, α-helical CRF9-41 (αCRF), in a randomized order. Brain sections were processed for c-fos by immunohistochemistry followed by image analysis at defined levels of the brain. CARTp (1µg, 4th icv) induced a robust c-fos response in the nucleus of the solitary tract (NTS) and area postrema (AP), whereas, no c-fos could be detected in the parabrachial nucleus (PBN), the paraventricular nucleus of the hypothalamus (PVN) or the arcuate nucleus of the hypothalamus (ARC). The c-fos expression in the structures of the dorsal vagal complex (DVC) was completely blocked by pre-treatment with the CRF antagonist, which did not by itself induce c-fos at any examined level. After CARTp and αCRF in combination, there was a tendency towards an increased c-fos response in the ARC. We conclude that CARTp activates cells of the area postrema and NTS via a downstream, CRF-dependent mechanism.

Ethanol induced hepatic mitochondrial dysfunction is attenuated by all trans retinoic acid supplementation.

AIMS: Alcoholics have reduced vitamin A levels in serum since vitamin A and ethanol share the same metabolic pathway. Vitamin A supplementation has an additive effect on ethanol induced toxicity. Hence in this study, we assessed the impact of supplementation of all trans retinoic acid (ATRA), an active metabolite of vitamin A on ethanol induced disruptive alterations in liver mitochondria. METHODS: Male Sprague Dawley rats were grouped as follows: I: Control; II: Ethanol (4 g/kg b.wt./day); III: ATRA (100 µg/kg b.wt./day); and IV: Ethanol (4 g/kg b.wt./day)+ATRA (100 µg/kg b.wt./day). Duration of the experiment was 90 days, after which the animals were sacrificed for the study. The key enzymes of energy metabolism, reactive oxygen species, mitochondrial membrane potential and hepatic mRNA expressions of Bax, Bcl-2, c-fos and c-jun were assessed. KEY FINDINGS: Ethanol administration increased the reactive oxygen species generation in mitochondria. It also decreased the activities of the enzymes of citric acid cycle and oxidative phosphorylation. ATP content and mitochondrial membrane potential were decreased and cytosolic cytochrome c was increased consequently enhancing apoptosis. All these alterations were altered significantly on ATRA supplementation along with ethanol. These results were reinforced by our histopathological studies. SIGNIFICANCE: ATRA supplementation to ethanol fed rats, led to reduction in oxidative stress, decreased calcium overload in the matrix and increased mitochondrial membrane potential, which might have altered the mitochondrial energy metabolism and elevated ATP production thereby reducing the apoptotic alterations. Hence ATRA supplementation seemed to be an effective intervention against alcohol induced mitochondrial dysfunction.

Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models.

Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well.

Brain region-specific reduction in c-Fos expression associated with an anxiolytic effect of yokukansan in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: A traditional Japanese (Kampo) medicine, yokukansan, has long been used to treat neurosis, insomnia, and night crying and irritability in children. Recently, this medicine has reported to improve the behavioral and psychological symptoms of dementia that often become problematic in patients with Alzheimer's disease and other forms of dementia. AIM OF THE STUDY: Several animal studies have reported that yokukansan has an anxiolytic effect. However, the underlying mechanisms are not yet understood. In the present study, we investigated the effects in rats of single and repeated administrations of yokukansan on anxiety-like behaviors, stress responses, and the brain regions involved. MATERIALS AND METHODS: Yokukansan dissolved in water (100 or 300 mg/kg) was administered orally to F344/N male rats 1h before each test or for two weeks before the tests began. Locomotor activity and anxiety-related behavior in the open-field test and the elevated plus-maze test, serum corticosterone levels, and restraint stress-induced c-Fos expression in various brain regions as a marker of neuronal activation were evaluated in both the vehicle-treated and yokukansan-treated rats. RESULTS: A single administration of yokukansan had no effect on locomotor activity or anxiety-like behavior; however, repeated administration decreased anxiety-like behavior in a dose-dependent manner. Neither single nor repeated administration of yokukansan had an effect on the basal or stress-induced levels of serum corticosterone. For c-Fos expression, restraint stress increased the number of c-Fos-positive cells in the subdivisions of the prefrontal cortex, amygdala, and hypothalamus. Repeated administration of yokukansan decreased the stress-induced c-Fos expression in the prelimbic cortex and the basolateral and medial amygdaloid nuclei. CONCLUSIONS: The present study indicates that repeated oral administration of yokukansan has an anxiolytic effect and that this effect may be associated with attenuated neuronal activity in the medial prefrontal cortex and amygdala.

Neuron discharge and c-Fos expression in the nucleus of the solitary tract following electroacupuncture at acupoints of the Yangming Stomach Meridian of Foot.

The nucleus of the solitary tract (nucleus tractus solitarii; NTS) is a primary center for both visceral afferents and somatic afferents. Previous experiments have demonstrated that the NTS is closely connected to the stomach and acupoints in the Yangming Stomach Meridian of Foot (ST Meridian). In this study, extracellular recording and immunochemistry methods were used to analyze the discharge of neurons and c-Fos protein expression in the NTS following acupuncture at different acupoints and a nonacupoint. A total of 104 discharging neurons were detected in the NTS of 52 rats, of which 86 provided complete data. After acupuncture at Sibai (ST 2), Zusanli (ST 36), Neiting (ST 44), Quanliao (SI 18), and the nonacupoint, the neuron response rate in the NTS was 65.12%, 51.16%, 46.51%, 34.88% and 31.40% respectively. For neuron response rate, there was a significant difference among Sibai (ST 2), Zusanli (ST 36), Neiting (ST 44), Quanliao (SI 18), and the nonacupoint (p < 0.01 or p < 0.05). In the other 48 rats, the number of c-Fos immunoreactive neurons in the NTS by electroacupuncture (EA) at Sibai (ST 2) group was significantly higher than that EA at other acupoints and the nonacupoint (p < 0.05 or p < 0.01). EA at both Zusanli (ST 36) and Neiting (ST 44) increased c-Fos immunoreactive neurons significantly over EA at Quanliao (SI 18) and the nonacupoint (p < 0.05 or p < 0.01), while there was no difference between EA at Quanliao (SI 18) and the nonacupoint group (p > 0.05). The experiments demonstrated that the afferent convergence in NTS are different by body surface points stimulus, which suggests that the NTS might be a primary center in the central nervous system receiving acupoints stimulus from the ST Meridian.

Dynein light chain LC8 inhibits osteoclast differentiation and prevents bone loss in mice.

NF-κB is one of the key transcription factors activated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. The 8-kDa dynein L chain (LC8) was previously identified as a novel NF-κB regulator. However, its physiological role as an NF-κB inhibitor remains elusive. In this study, we showed the inhibitory role of LC8 in RANKL-induced osteoclastogenesis and signaling pathways and its protective role in osteolytic animal models. LC8 suppressed RANKL-induced osteoclast differentiation, actin ring formation, and osteoclastic bone resorption. LC8 inhibited RANKL-induced phosphorylation and subsequent degradation of IκBα, the expression of c-Fos, and the consequent activation of NFATc1, which is a pivotal determinant of osteoclastogenesis. LC8 also inhibited RANKL-induced activation of JNK and ERK. LC8-transgenic mice exhibited a mild osteopetrotic phenotype. Moreover, LC8 inhibited inflammation-induced bone erosion and protected against ovariectomy-induced bone loss in mice. Thus, our results suggest that LC8 inhibits osteoclast differentiation by regulating NF-κB and MAPK pathways and provide the molecular basis of a new strategy for treating osteoporosis and other bone diseases.

Orexin-A suppresses postischemic glucose intolerance and neuronal damage through hypothalamic brain-derived neurotrophic factor.

Orexin-A (a glucose-sensing neuropeptide in the hypothalamus) and brain-derived neurotrophic factor (BDNF; a member of the neurotrophin family) play roles in many physiologic functions, including regulation of glucose metabolism. We previously showed that the development of postischemic glucose intolerance is one of the triggers of ischemic neuronal damage. The aim of this study was to determine whether there was an interaction between orexin-A and BDNF functions in the hypothalamus after cerebral ischemic stress. Male ddY mice were subjected to 2 hours of middle cerebral artery occlusion (MCAO). Neuronal damage was estimated by histologic and behavioral analyses. Expression of protein levels was analyzed by Western blot. Small interfering RNA directed BDNF, orexin-A, and SB334867 [N-(2-methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea; a specific orexin-1 receptor antagonist] were administered directly into the hypothalamus. The level of hypothalamic orexin-A, detected by immunohistochemistry, was decreased on day 1 after MCAO. Intrahypothalamic administration of orexin-A (1 or 5 pmol/mouse) significantly and dose-dependently suppressed the development of postischemic glucose intolerance on day 1 and development of neuronal damage on day 3. The MCAO-induced decrease in insulin receptor levels in the liver and skeletal muscle on day 1 was recovered to control levels by orexin-A, and this effect of orexin-A was reversed by the administration of SB334867 as well as by hypothalamic BDNF knockdown. These results suggest that suppression of postischemic glucose intolerance by orexin-A assists in the prevention of cerebral ischemic neuronal damage. In addition, hypothalamic BDNF may play an important role in this effect of orexin-A.

[Detection of c-fos expression in animal brain in a pilocarpine model of temporal lobe epilepsy].

The dynamics of the involvement of different brain structures in a pathological process is very important for decoding the mechanisms of temporal lobe epilepsy. In this work, the experimental model of temporal lobe epilepsy induced by lithium chloride and pilocarpine was used. The method of immunochemical detection of the immediate early gene c-fos was used as an indicator of functioning neurons in the brain. The c-fos expression was determined at different time points (30, 60 and 90 min) after the pilocarpine injection. An increase in the c-fos expression was observed in neuronal populations during the development of the status epilepticus, the time and degree of involvement of different brain structures being different. The expression of c-fos was first observed in the piriform cortex, the olfactory tubercle, thalamic nuclei, lateral habenular nuclei, and the caudate putamen. Then the hippocampus, the septal formation, the amygdala, and basal ganglia were involved in the activation process. In the hypothalamic areas, c-fos expression was observed latest. These data contribute to understanding the mechanisms of temporal lobe epilepsy and searching for the ways of its therapy.

Orexinergic signaling mediates light-induced neuronal activation in the dorsal raphe nucleus.

Seasonal affective disorder (SAD), a major depressive disorder recurring in the fall and winter, is caused by the reduction of light in the environment, and its depressive symptoms can be alleviated by bright light therapy. Both circadian and monoaminergic systems have been implicated in the etiology of SAD. However, the underlying neural pathways through which light regulates mood are not well understood. The present study utilized a diurnal rodent model, Arvicanthis niloticus, to explore the neural pathways mediating the effects of light on brain regions involved in mood regulation. Animals kept in constant darkness received light exposure in early subjective day, the time when light therapy is usually applied. The time course of neural activity following light exposure was assessed using Fos protein as a marker in the following brain regions/cells: the suprachiasmatic nucleus (SCN), orexin neurons in the perifornical-lateral hypothalamic area (PF-LHA) and the dorsal raphe nucleus (DRN). A light-induced increase in Fos expression was observed in orexin neurons and the DRN, but not in the SCN. As the DRN is densely innervated by orexinergic inputs, the involvement of orexinergic signaling in mediating the effects of light on the DRN was tested in the second experiment. The animals were injected with the selective orexin receptor type 1 (OXR1) antagonist SB-334867 prior to the light exposure. The treatment of SB-334867 significantly inhibited the Fos induction in the DRN. The results collectively point to the role of orexin neurons in mediating the effects of light on the mood-regulating monoaminergic areas, suggesting an orexinergic pathway that underlies light-dependent mood fluctuation and the beneficial effects of light therapy.

The differential profiles of withdrawal symptoms induced by morphine and beta-endorphin administered intracerebroventricularly in mice.

In the present study, withdrawal symptoms induced by morphine or ß-endorphin administered intracerebroventricularly (i.c.v.) were compared in ICR mice. Naloxone (10mg/kg) was post-treated intraperitoneally (i.p.) 3h after either a single or repeated (1 time/day for 3 days) i.c.v. injections with opioids. Withdrawal symptoms such as jumping frequency, diarrhea, weight loss, rearing, penile licking and paw tremor were observed for 30 min immediately after naloxone treatment. Withdrawal symptoms (jumping, diarrhea, weight loss, rearing, penile licking and paw tremor) observed in the group treated with morphine was persistently increased during 3 days. On the other hand, withdrawal symptoms such as diarrhea, weight loss and rearing in ß-endorphin-treated group were increased after a single injection with ß-endorphin, but gradually decreased after the repeated injection. Furthermore, no jumping behavior, penile licking and paw tremor in ß-endorphin-treated group were observed throughout the whole period of time. In addition, the hypothalamic changes of several signal molecules such as pERK, pCaMK-IIα, c-FOS and pCREB expression were observed during the presence or absence of withdrawal responses induced by morphine or ß-endorphin administered once or repeatedly. Both hypothalamic pCaMK-IIα and c-FOS expressions were increased by naloxone treatment in acutely administered morphine group, whereas only pCaMK-IIα expression was elevated by naloxone treatment in repeatedly administered morphine group. In contrast with the findings in morphine-treated group, only pCaMK-IIα expression was decreased by naloxone treatment in repeatedly administered ß-endorphin group. Our results suggest that profiles of the withdrawal symptoms induced by morphine and ß-endorphin administered supraspinally appear to be differentially regulated. The pCaMK-IIα and the c-FOS protein expression may play important roles for the regulation of naloxone-precipitated withdrawal symptoms such as jumping, diarrhea, weight loss, rearing, penile licking and paw tremor induced by morphine-treated group, whereas the phosphorylation of hypothalamic pCaMK-IIα appears to be involved only in the regulation of naloxone-precipitated withdrawal symptoms such as diarrhea, weight loss and rearing in ß-endorphin-treated group.

Pontomedullary and hypothalamic distribution of Fos-like immunoreactive neurons after acute exercise in rats.

During exercise, intense brain activity orchestrates an increase in muscle tension. Additionally, there is an increase in cardiac output and ventilation to compensate the increased metabolic demand of muscle activity and to facilitate the removal of CO(2) from and the delivery of O(2) to tissues. Here we tested the hypothesis that a subset of pontomedullary and hypothalamic neurons could be activated during dynamic acute exercise. Male Wistar rats (250-350 g) were divided into an exercise group (n=12) that ran on a treadmill and a no-exercise group (n=7). Immunohistochemistry of pontomedullary and hypothalamic sections to identify activation (c-Fos expression) of cardiorespiratory areas showed that the no-exercise rats exhibited minimal Fos expression. In contrast, there was intense activation of the nucleus of the solitary tract, the ventrolateral medulla (including the presumed central chemoreceptor neurons in the retrotrapezoid/parafacial region), the lateral parabrachial nucleus, the Kölliker-Fuse region, the perifornical region, which includes the perifornical area and the lateral hypothalamus, the dorsal medial hypothalamus, and the paraventricular nucleus of the hypothalamus after running exercise. Additionally, we observed Fos immunoreactivity in catecholaminergic neurons within the ventrolateral medulla (C1 region) without Fos expression in the A2, A5 and A7 neurons. In summary, we show for the first time that after acute exercise there is an intense activation of brain areas crucial for cardiorespiratory control. Possible involvement of the central command mechanism should be considered. Our results suggest whole brain-specific mobilization to correct and compensate the homeostatic changes produced by acute exercise.