Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections.

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes' expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called 'Quanty-cFOS', with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells.

Stroboscopic lighting with intensity synchronized to rotation velocity alleviates motion sickness gastrointestinal symptoms and motor disorders in rats.

Motion sickness (MS) is caused by mismatch between conflicted motion perception produced by motion challenges and expected "internal model" of integrated motion sensory pattern formed under normal condition in the brain. Stroboscopic light could reduce MS nausea symptom via increasing fixation ability for gaze stabilization to reduce visuo-vestibular confliction triggered by distorted vision during locomotion. This study tried to clarify whether MS induced by passive motion could be alleviated by stroboscopic light with emitting rate and intensity synchronized to acceleration-deceleration phase of motion. We observed synchronized and unsynchronized stroboscopic light (SSL: 6 cycle/min; uSSL: 2, 4, and 8 cycle/min) on MS-related gastrointestinal symptoms (conditioned gaping and defecation responses), motor disorders (hypoactivity and balance disturbance), and central Fos protein expression in rats receiving Ferris wheel-like rotation (6 cycle/min). The effects of color temperature and peak light intensity were also examined. We found that SSL (6 cycle/min) significantly reduced rotation-induced conditioned gaping and defecation responses and alleviated rotation-induced decline in spontaneous locomotion activity and disruption in balance beam performance. The efficacy of SSL against MS behavioral responses was affected by peak light intensity but not color temperature. The uSSL (4 and 8 cycle/min) only released defecation but less efficiently than SSL, while uSSL (2 cycle/min) showed no beneficial effect in MS animals. SSL but not uSSL inhibited Fos protein expression in the caudal vestibular nucleus, the nucleus of solitary tract, the parabrachial nucleus, the central nucleus of amygdala, and the paraventricular nucleus of hypothalamus, while uSSL (4 and 8 cycle/min) only decreased Fos expression in the paraventricular nucleus of hypothalamus. These results suggested that stroboscopic light synchronized to motion pattern might alleviate MS gastrointestinal symptoms and motor disorders and inhibit vestibular-autonomic pathways. Our study supports the utilization of motion-synchronous stroboscopic light as a potential countermeasure against MS under abnormal motion condition in future.

Evaluation of Two Injection Techniques in Combination with the Local Anesthetics Lidocaine and Mepivacaine for Piglets Undergoing Surgical Castration.

The present study evaluated the effects of two injection techniques in combination with lidocaine or mepivacaine for piglets undergoing castration. To improve ease of use, a cannula with side holes (one-step fenestrated (F)) was invented to simultaneously deliver a local anesthetic into the testis and scrotum and was compared to a two-step injection technique. The distribution of a lidocaine/contrast agent mixture using the two methods was examined using computed tomography. Piglets were randomly divided into treatment groups: handling, castration without pain relief and castration after lidocaine or mepivacaine injection using the one-step F or two-step method. Acute physiological responses to noxious stimuli were evaluated by measuring the mean arterial blood pressure (MAP), heart rate (HR) and nocifensive movements. Fos protein expression in the spinal dorsal cord was semi-quantitatively analyzed. Both injection techniques achieved similar distribution patterns. The one-step F method was faster and easier. Injection was not associated with significant changes in MAP or HR, but Mepi1 and NaCl elicited significantly increased nocifensive movements. Both techniques significantly reduced MAP and nocifensive movements when the spermatic cords were cut, regardless of the local anesthetic type. Compared to NaCl, only the lidocaine treatments significantly reduced HR during skin incision. Lido2 significantly reduced Fos protein expression.

Brain adaptation to acute stress: effect of time, social buffering, and nicotinic cholinergic system.

Both social behavior and stress responses rely on the activity of the prefrontal cortex (PFC) and basolateral nucleus of the amygdala (BLA) and on cholinergic transmission. We previously showed in adult C57BL/6J (B6) mice that social interaction has a buffering effect on stress-related prefrontal activity, depending on the ß2-/- cholinergic nicotinic receptors (nAChRs, ß2-/- mice). The latency for this buffer to emerge being short, we question here whether the associated brain plasticity, as reflected by regional c-fos protein quantification and PFC-BLA functional connectivity, is modulated by time. Overall, we show that time normalized the stress-induced PFC hyperactivation in B6 mice and PFC hypo-activation in ß2-/- mice, with no effect on BLA. It also triggered a multitude of functional links between PFC subareas, and between PFC and BLA in B6 mice but not ß2-/- mice, showing a central role of nAChRs in this plasticity. Coupled with social interaction and time, stress led to novel and drastic diminution of functional connectivity within the PFC in both genotypes. Thus, time, emotional state, and social behavior induced dissociated effects on PFC and BLA activity and important cortico-cortical reorganizations. Both activity and plasticity were under the control of the ß2-nAChRs.

Neuromedin U modulates neuronal excitability in rat hippocampal slices.

Neuromedin U (NMU) is a neuropeptide that was initially isolated from the porcine spinal cord and later from several species. Although NMU receptors exist in the CA1 region of the hippocampus, the role of NMU in hippocampal synaptic transmission remains unknown. In the present study, we demonstrated that the colocalization ratio of NMU type 1 (NMUR1) or type 2 (NMUR2) receptors was higher with neuronal nuclei (a neuronal marker) than with glial fibrillary acidic protein (an astrocyte marker) in the CA1 region of rats. Moreover, we revealed that the bath application of NMU (1 µM) enhanced extracellular field excitatory postsynaptic potentials at Schaffer collateral-CA1 synapses in rat hippocampal slices (+28.9 ± 1.3%; P < 0.05). After extracellular recordings, we examined the pattern of neuronal activation induced by NMU using c-Fos immunohistochemistry (Fos-IR). Histological analyses revealed that NMU increased Fos-IR in the CA1 region, but reduced the proportion of Fos-IR colocalized with glutamic acid decarboxylase (a GABA neuron marker). These results suggest that the activation of NMU receptors contributes to GABAergic neuronal activity in the CA1 region of the hippocampus.

Ameliorating effects of histamine H3 receptor antagonist E177 on acute pentylenetetrazole-induced memory impairments in rats.

Histamine H3 receptors (H3Rs) are involved in several neuropsychiatric diseases including epilepsy. Therefore, the effects of H3R antagonist E177 (5 and 10 mg/kg, intraperitoneal (i.p.)) were evaluated on acute pentylenetetrazole (PTZ)-induced memory impairments, oxidative stress levels (glutathione (GSH), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD)), various brain neurotransmitters (histamine (HA), acetylcholine (ACh), γ-aminobutyric acid (GABA)), and glutamate (Glu), acetylcholine esterase (AChE) activity, and c-fos protein expression in rats. E177 (5 and 10 mg/kg, i.p.) significantly prolonged step-through latency (STL) time in single-trial passive avoidance paradigm (STPAP), and shortened transfer latency time (TLT) in elevated plus maze paradigm (EPMP) (all P < 0.05). Moreover, and in the hippocampus of PTZ-treated animals, E177 mitigated abnormal levels of AChE activity, ACh and HA (all P < 0.05), but failed to modify brain levels of GABA and Glu. Furthermore, E177 alleviated hippocampal oxidative stress by significantly decreasing the elevated levels of MDA, and increasing the abnormally decreased level of GSH (all P < 0.05). Furthermore, E177 reduced elevated levels of hippocampal c-fos protein expression in hippocampal tissues of PTZ-treated animals (all P < 0.05). The observed results propose the potential of H3R antagonist E177 with an added advantage of avoiding cognitive impairment, emphasizing the H3Rs as a prospective target for future pharmacological management of epilepsy with associated memory impairments.

Low Fos expression in newly generated neurons of the main and accessory olfactory bulb following single maternal separation.

The main and accessory olfactory bulbs (MOB and AOB) are unique in that they produce new neurons throughout adulthood. Despite the recent knowledge about the involvement of postnatally generated cells in several aspects of olfaction, the functional role of these neurons is still not sufficiently understood. The function of newly generated olfactory bulb neurons is primarily investigated in relation to activities related to smell. Stress-induced activation of new olfactory neurons has not yet been studied. Thus, our work was aimed to investigate whether a stressful event, such as maternal separation (MS) can induce Fos expression in postnatally-born neurons in the MOB and AOB. Rat pups were exposed to single maternal separation (SMS) for 2 h at the postnatal days: P7, P14, and P21. Quantification of immunohistochemically labeled Fos + cells revealed that exposure to SMS in different age stages during the first postnatal month stimulates activity in cells of individual MOB/AOB layers in an age-dependent manner. In order to find out whether newly generated cells in the MOB/AOB could express Fos protein as a response to SMS, newborn rats were administrated with the marker of proliferation, bromodeoxyuridine (BrdU) at P0, and three weeks later (at P21) colocalization of Fos and BrdU in the neurons of the MOB and AOB was assessed. Quantitative analysis of BrdU/Fos double-labeled cells showed that Fos is expressed only in a small number of postnatally generated cells within the MOB/AOB. Our results indicate that postnatally generated MOB/AOB neurons are less sensitive to stress caused by MS than preexisting ones. LAY SUMMARY Our results showed that single maternal separation (SMS) is a stressful event that in age-dependent manner stimulates cellular activity in the main and accessory olfactory bulb (AOB) - the structures dedicated to odor information processing. The low level of Fos expression in newborn neurons of the main and accessory bulb indicates that postnatally generated cells are less sensitive to neonatal stress than preexisting neurons.

Cyclosomatostatin-induced catalepsy in aged rats: Specific change of brain c-Fos protein expression in the lateral entorhinal cortex.

Aging represents the largest risk factor for developing Parkinson's disease (PD); another salient feature of this disorder is a decreased brain levels of somatostatin. Recently, in aged Wistar rats, we simulated the central somatostatinergic deficiency by intracerebroventricular injections of a somatostatin antagonist, cyclosomatostatin (cSST). The treated animals displayed catalepsy, a state that resembles the extrapyramidal signs of Parkinson's disease; young animals were insensitive to cSST. The neuroanatomical substrates responsible for the increased cataleptogenic activity of cSST in aged animals, are currently unknown. To study this issue, we assessed the cSST effect on brain c-Fos-protein expression in aged and young rats; thirty three brain regions were examined. cSST was employed at the dose cataleptogenic for aged animals and non-cataleptogenic for young ones. c-Fos expression patterns in the 'cataleptic' and 'non-cataleptic' animals were very similar, with the only distinction being a decrease in the c-Fos expression in the aged lateral entorhinal cortex (LEntCx). This decrease was not observed when the cSST-induced cataleptic response was inhibited by administration of diphenhydramine and nicotine. Thus, the development of catalepsy in the aged Wistar rats appeared to be associated with a hypoactivation of the LEntCx; possibly, there exists a mechanistic link between the LEntCx hypoactivation and increased susceptibility of aged rats to catalepsy. Apparently, these findings may provide novel insight into the link between mechanisms of parkinsonian motor disorders and aging.

Antagonism of Histamine H3 receptors Alleviates Pentylenetetrazole-Induced Kindling and Associated Memory Deficits by Mitigating Oxidative Stress, Central Neurotransmitters, and c-Fos Protein Expression in Rats.

Histamine H3 receptors (H3Rs) are involved in several neuropsychiatric diseases including epilepsy. Therefore, the effects of H3R antagonist E177 (5 and 10 mg/kg, intraperitoneal (i.p.)) were evaluated on the course of kindling development, kindling-induced memory deficit, oxidative stress levels (glutathione (GSH), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD)), various brain neurotransmitters (histamine (HA), acetylcholine (ACh), γ-aminobutyric acid (GABA)), and glutamate (GLU), acetylcholine esterase (AChE) activity, and c-Fos protein expression in pentylenetetrazole (PTZ, 40 mg/kg) kindled rats. E177 (5 and 10 mg/kg, i.p.) significantly decreased seizure score, increased step-through latency (STL) time in inhibitory avoidance paradigm, and decreased transfer latency time (TLT) in elevated plus maze (all P < 0.05). Moreover, E177 mitigated oxidative stress by significantly increasing GSH, CAT, and SOD, and decreasing the abnormal level of MDA (all P < 0.05). Furthermore, E177 attenuated elevated levels of hippocampal AChE, GLU, and c-Fos protein expression, whereas the decreased hippocampal levels of HA and ACh were modulated in PTZ-kindled animals (all P < 0.05). The findings suggest the potential of H3R antagonist E177 as adjuvant to antiepileptic drugs with an added advantage of preventing cognitive impairment, highlighting the H3Rs as a potential target for the therapeutic management of epilepsy with accompanied memory deficits.

Differential effects of acute and chronic treatment with the flavonoid chrysin on anxiety-like behavior and Fos immunoreactivity in the lateral septal nucleus in rats.

The aim of this study was to compare the effects of acute (a single injection) and chronic (21 consecutive days) treatments with chrysin 2, 4, and 8 µmol kg-1 on anxiety-like behavior and Fos immunoreactivity in the lateral septum nucleus (LSN), a structure that is involved in the regulation of anxiety, in male Wistar rats. These effects were compared with the clinically effective anxiolytic diazepam 7 µmol kg-1. The results showed that acute, but not chronic treatment, with 4 µmol kg-1 chrysin exerted anxiolytic- and anti- depressant-like effects with these effects being similar to that of diazepam. Also, none of the above-mentioned treatments did alter Fos immunoreactivity in the LSN, but a tendency towards the reduction of this variable was detected with chrysin 4 µmol kg-1 and diazepam 7 µmol kg-1. Altogether, results suggest that chrysin exerts anxiolytic-like effects, however, it can produce pharmacological tolerance after repeated use, similar to benzodiazepines.

Expression of estrogen receptor α variants and c-Fos in rat mammary gland and tumors.

Breast cancer is currently the leading cause of cancer death among women worldwide. AP-1 (c-Fos/c-Jun) is associated with proliferation and survival, while cytoplasmic c-Fos activates phospholipid synthesis in cells induced to differentiate or grow. Estrogen receptor α 46 (ERα46) is a splice variant of full-length ERα66 and it is known that it has an inhibitory role in cancer cell growth. We investigated c-Fos localization, its relationship to AP-1, the non genomic pathway of phospho-Tyr537-ERα66, as well as ERα46 and ERα66 isoforms in rat mammary gland development and carcinogenic transformation, and in mammary tumors. Female rats were injected: a) saline solution (Control mammary gland, CMG) or b) N-Nitroso-N-methyl urea (NMU), and samples were taken at 60, 90, 120 and 150 days of life. In addition, we analyzed hormone-dependent (HD) and independent (HI) tumors in ovariectomized rats, and intact tumors (IT) in non-ovariectomized ones. Our results show that, in CMG, nuclear c-Fos and proliferation decreased with age, AP-1 content was low, and nuclear ERα46/ERα66 ratio was higher than 1. In NMU, nuclear c-Fos and proliferation increased with carcinogenic transformation, AP-1 content was high, and nuclear ERα46/ERα66 was below 1. As tumor grade increased, proliferation, nuclear c-Fos and AP-1 expression were negatively associated to nuclear ERα46/ERα66 in IT. In HD, nuclear ERα46/ERα66, nuclear c-Fos expression, AP-1 levels and proliferation were lower than in HI, whose growth is estrogen-independent. Phospho-Tyr537-ERα66 content and ERK1/2 activation were associated with AP-1 levels and cell proliferation. Collectively, our findings support the notion that variant detection and ERα46/ERα66 ratio could shed light on the role of ERα isoforms in mammary gland transformation and the behavior of ERα positive mammary tumors.

Pattern of neural activation following yohimbine-induced reinstatement of alcohol seeking in rats.

Alcohol use disorders represent an extensive socioeconomic burden, yet effective treatment options are suboptimal. A major hurdle in treating alcohol use disorders is the high rate of relapse. Stress is a major factor that promotes relapse in abstinent drug users; therefore, understanding neural mechanisms that underpin the effects of stress on alcohol seeking is critical. In rodent models of stress-induced relapse, the α2 -adrenoceptor antagonist, yohimbine, is a widely used chemical stressor to elicit reinstatement of drug/alcohol seeking. However, the exact mechanism how yohimbine precipitates reinstatement of alcohol seeking and the pattern of neural activation associated with yohimbine-induced reinstatement is poorly understood. Therefore, we counted Fos-protein positive nuclei across 42 brain regions in alcohol-experienced alcohol preferring rats that received either yohimbine in the home-cage (1 mg/kg i.p.) or following yohimbine-induced reinstatement of alcohol seeking. The number of Fos-protein positive nuclei was increased in the prefrontal cortex and extended amygdala after home-cage yohimbine compared to naïve- and vehicle-treated rats. Yohimbine-induced reinstatement increased the number of Fos-protein expressing nuclei in multiple other regions including the thalamus, hypothalamus and hippocampus. We then examined inter-regional correlations in Fos-protein expression for all 42 brain regions, which showed Fos expression was more strongly positively correlated following yohimbine-induced reinstatement of alcohol seeking, compared to home-cage yohimbine. These data suggest low-dose yohimbine in a non-drug-associated context activates stress/impulsivity centres within the brain, whereas yohimbine in the drug-associated context recruits additional brain regions to drive alcohol seeking.

The effect of prenatal and postnatal caffeine exposure on pentylentetrazole induced seizures in the non-epileptic and epileptic offsprings.

Caffeine, a central nervous system stimulant, has been reported to modulate seizure activity in various studies. In this study the effects of caffeine exposure on the pentylenetetrazole (PTZ) induced seizure thresholds and seizure stages in the Wistar and genetic absence epilepsy model offsprings were examined. Adult female and male Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS) consumed caffeine dissolved in water (0.3 g/L) before conception, during the gestational periods and lactation period whereas control groups of each strain received tap water. All offsprings at postnatal day 30 (PN30) subjected to 70 mg/kg of PTZ were evaluated in terms of overall seizure stages, the latency to the first generalized seizure and the c-Fos protein activity in the brain regions of somatosensorial cortex (SSCx), reticular thalamic nucleus (Rt), ventrobasal thalamus (VB), centromedial nucleus (CM) and lateral geniculate nucleus (LGN). The Wistar caffeine group had significantly shorter latency to the first generalized seizure (1.53 ±â€¯0.49 min) comparing to the Wistar control offsprings (3.40 ±â€¯0.68 min). GAERS caffeine group (6.52 ±â€¯2.48 min) showed significantly longer latency comparing to Wistar caffeine group (1.53 ±â€¯0.49 min). Although statistically not significant, GAERS caffeine group showed a longer latency comparing to the GAERS control group (4.71 ±â€¯1.82 min). In all regions of SSCx, Rt, VB, CM and LGN, GAERS caffeine group had lower c-Fos protein expression comparing to the GAERS control group (p < 0.05). Wistar caffeine rats had lower expression of c-Fos protein comparing to the Wistar control group only in SSCx. In CM, GAERS rats expressed lower c-Fos protein comparing to the Wistar control (p < 0.05). In conclusion differential effects of caffeine in the seizure modulation may involve c-Fos protein activity-dependent protection mechanisms.

Mechanism Underlying Organophosphate Paraoxon-Induced Kinetic Tremor.

Organophosphates (OPs) inhibit cholinesterase and hyperactivate the acetylcholinergic nervous system in the brain, causing motor disorders (e.g., tremor and seizures). Here, we performed behavioral and immunohistochemical studies in mice and rats to investigate the tremorgenic mechanism of paraoxon, an active metabolite of parathion. Treating animals with paraoxon (0.15-0.6 mg/kg, i.p.) elicited kinetic tremor in a dose-dependent manner. Expressional analysis of Fos protein, a biomarker of neural excitation, revealed that a tremorgenic dose of paraoxon (0.6 mg/kg) significantly and region-specifically elevated Fos expression in the cerebral cortex (e.g., sensory cortex), hippocampal CA1, globus pallidus, medial habenula, and inferior olive (IO) among 48 brain regions examined. A moderate increase in Fos expression was also observed in the dorsolateral striatum while the change was not statistically significant. Paraoxon-induced tremor was inhibited by the nicotinic acetylcholine (nACh) receptor antagonist mecamylamine (MEC), but not affected by the muscarinic acetylcholine receptor antagonist trihexyphenidyl (THP). In addition, paraoxon-induced Fos expression in the IO was also antagonized by MEC, but not by THP, and lesioning of the IO markedly suppressed tremorgenic action of paraoxon. The present results suggest that OPs elicit kinetic tremor at least partly by activating IO neurons via nACh receptors.

The effect of acute or repeated stress on the corticotropin releasing factor system in the CRH-IRES-Cre mouse: A validation study.

Corticotropin releasing factor (CRF) is a key component of stress responsivity, modulating related behaviors including anxiety and reward. Difficulties identifying CRF neurons, using traditional approaches including immunohistochemistry, has led to the development of a number of transgenic CRF reporter mice. The Crh-IRES-Cre::Ai14 (tdTomato) reporter mouse is increasing in popularity as a useful tool to assess the localization, connectivity and function of CRF neurons in various stress-related behaviors. However, without proper characterization of reporter expression, the in vivo and in vitro manifestations resulting from the manipulation of these cells must be interpreted with caution. Here we mapped the distribution of tdTomato-expressing CRF cells throughout the rostro-caudal extent of the Crh-IRES-Cre::Ai14 mouse brain. To determine if reporter expression faithfully reproduced native CRF expression, we assessed the colocalization of CRF expression with tdTomato reporter expression across several brain regions. Good concordance was observed in the extended amygdala and paraventricular nucleus of the hypothalamus (PVN), while discrepancies were observed within the lateral hypothalamus and hippocampus. Finally, we examined the activation of CRF neurons in Crh-IRES-Cre::Ai14 mice in response to different types of stressors using Fos immunohistochemistry. Acute psychological (swim) and pharmacological (yohimbine) stress stimulated Fos-protein expression in PVN CRF neurons. Interestingly though, exposure to four daily restraint stress sessions followed by a novel acute stressor did not further recruit CRF neurons across any brain region examined. Our results highlight the importance of thoroughly characterizing reporter mice before use and suggest that acute versus repeated stress may differentially impact the CRF system. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

Comparison of Improvement Effects and Its Mechanism of Different Doses of Total Alkaloids from Aconitum racemulosum on CIA Model Rats Based on Information Entropy Theory / 中国药房

OBJECTIVE： To investigate the effects of different doses of total alkaloids from Aconitum racemulosum （ARTA） on serum inflammation factors and FOS protein expression in synovial tissue of joint in collagen-induced arthritis （CIA） model rats， and to investigate its potential mechanism of anti-rheumatoid arthritis （RA）. METHODS： Male SD rats were randomly divided into blank group， model group， positive group （Compound dexamethasone acetate ointment， 0.2 g/kg）， ARTA low-dose， medium-dose and high-dose groups （56.26， 112.50， 225.00 mg/kg， by the weight of ARTA in the extract）， with 10 rats in each group. Except for blank group， other groups were given subcutaneous injection of Bovine collagen Ⅱ emulsified with incomplete Freund’s adjuvant into the left foot to establish CIA model； the left foot were smeared with relevant medicine from the day of modeling. Blank group and model group were smeared with constant volume of 65％ ethanol， 3 times a day， for consecutive 28 days. On the 7th， 14th， 21st and 28th day of administration， the thickness of left hind toe was measured with vernier caliper， and the degree of foot swelling was calculated. The serum contents of IL-1β， IL-6 and TNF-α in rats were measured by ELISA after last administration. The expression of FOS protein in synovial tissue was determined by immunohistochemical method [expressed by HIS]. The comprehensive score was conculated by entropy weight method. Effects of each dosage on above indexes of CIA model rats were evaluated with the comprehensive score. RESULTS： Compared with blank group， the degree of foot swelling， serum content of inflammatory factors and HIS value were increased significantly in model group （P＜0.05）. Compared with model group， the degree of foot swelling in each administration group， serum contents of IL-1β， IL-6 and TNF-α， HIS in positive group and ARTA high-dose group， serum contents of IL-6 and TNF-α in ARTA medium-dose group as well as serum content of TNF-α in ARTA low-dose group were decreased significantly（P＜0.05）. Comprehensive score of above indicators were 0.37（positive group）， 0.31（ARTA high-dose group）， 0.23（ARTA medium-dose group） and 0.09（ARTA low-dose group）. CONCLUSIONS： ARTA can improve CIA model rats， and the effect tends to increase with the increase of dose. Above effect may be associated with reducing serum content of inflammatory factors and inhibiting the expression of FOS protein in synovial tissue.

Aqueous extract of saffron administration along with amygdala deep brain stimulation promoted alleviation of symptoms in post-traumatic stress disorder (PTSD) in rats.

OBJECTIVE: Post-traumatic stress disorder (PTSD) as one of the most devastating kinds of anxiety disorders, is the consequence of a traumatic event. Crocus sativus L., commonly known as saffron have been traditionally used for treatment of stress and anxiety. In this study, we evaluated the effects of peripheral administration of saffron, along with deep brain stimulation (DBS) in a post-traumatic stress disorder (PTSD) model caused by contextual fear conditioning (electrical foot shock chamber) in male Wistar rats. MATERIALS AND METHODS: rats (220-250 g) were divided into 7 groups (n=8) and underwent stereotactic surgery for implantation of the electrodes in the right-baso lateral of the amygdala (BLA). After 7 days, some animals received the foot shock, followed by another 7-day treatment (DBS treatment or combination treatment by saffron 5 mg/kg (i.p)) then freezing behavior as a predicted response in the absence of the foot shock (re-exposure time) and general anxiety were measured using elevated plus maze test. Serum corticosterone level and amygdala c-Fos protein expression were assessed using ELISA and Western blot analysis, respectively. RESULTS: DBS treatment and the combination therapy of saffron (5 mg/kg (I.P)) with DBS significantly (p<0.001) increased serum corticosterone levels. Also both treatments could significantly (p<0.001) reduce c-Fos protein expression and freezing behaviors time. However, DBS treatment had no effect on the general anxiety in rats with PTSD. On the other hand, combination therapy significantly (p<0.001) reduced anxiety behavior in rats with PTSD. CONCLUSION: These results might show the potential of this combination therapy for treatment of treatment-resistant PTSD patients.

Effects of centrally administered glucagon-like peptide-2 on blood pressure and barosensitive neurons in spontaneously hypertensive rats.

The central administration of glucagon-like peptide-2 (GLP-2) decreases blood pressure in rats. In the present study, we investigated the hypotensive effects of GLP-2 using spontaneously hypertensive rats (SHRs), an animal model of hypertension. The central administration of GLP-2 (0.6 µg) decreased mean arterial pressure (MAP) in SHRs (-24.1 ±â€¯4.5%; P < 0.05), but not in normotensive Wistar-Kyoto (WKY) rats (-10.6 ±â€¯7.4%; P > 0.05), whereas GLP-2 (6 µg) decreased MAP in WKY rats (-23.5 ±â€¯4.2%; P < 0.05) and SHRs (-46.7 ±â€¯11.6%; P < 0.01) under anesthesia with urethane and α-chloralose. Histological analyses revealed that the central administration of GLP-2 (6 µg) induced Fos immunoreactivity (Fos-IR) in the hypothalamic and medullary areas in WKY rats and SHRs. However, the distribution of Fos-IR in GABAergic neurons in the rostral ventrolateral medulla (RVLM) differed between WKY rats and SHRs. GLP-2 directly modulated the excitability of RVLM neurons in brainstem slices from SHRs, but not WKY rats. These results suggest that neuronal activity through the activation of GLP-2 receptors in the RVLM contributes to lowering blood pressure in SHRs.

Non-competitive antagonists of NMDA and AMPA receptors decrease seizure-induced c-fos protein expression in the cerebellum and protect against seizure symptoms in adult rats.

The aim of the present study was to examine the role of ionotropic glutamate receptors in the cerebellum during generalized seizures. Epileptic neuronal activation was evaluated through the immunohistochemical detection of c-fos protein in the cerebellar cortex. Generalized seizures were precipitated by the intraperitoneal injection of 4-aminopyridine. The animals were pretreated with the NMDA receptor antagonists MK-801 (2 mg/kg), amantadine (50 mg/kg), and the AMPA receptor antagonist GYKI 52466 hydrochloride (50 mg/kg). Two hours after 4-aminopyridine injection, the number of c-fos immunostained cell nuclei was counted in serial immunohistochemical sections of the cerebellar vermis. The number of c-fos immunostained cell nuclei in the granular layer decreased significantly in animals pretreated with the glutamate receptor antagonists compared to the untreated animals having convulsion. We can conclude that mossy fiber stimulation exerts its seizure-generating action mainly through the ionotropic glutamate receptors of the mossy fiber synapses. Both NMDA and AMPA receptor antagonists are effective in reducing glutamate-mediated postsynaptic effects in the cerebellar cortex.

Evaluation of food intake and Fos expression in serotonergic neurons of raphe nuclei after intracerebroventricular injection of adrenaline in free-feeding rats.

Previous studies indicate that the modification of adrenergic neurotransmission in median raphe nucleus (MRN) enhances or removes an inhibitory influence on food intake, possibly serotonergic, due to a presence of serotonin-producing neurons in that nucleus. Therefore, the aim of this study is evaluated whether the activity of neurons in the MRN and dorsal raphe nucleus (DRN) are affected by intracerebroventricular injection of adrenaline (AD) in free-feeding rats. Male Wistar rats with guide cannulae chronically implanted in the lateral ventricle were injected with AD followed by evaluation of ingestive behavioral parameters. Behavior was monitored and the amount of food ingested was assessed. The highest dose (20 nmol) of AD was the most effective dose in increasing food intake. Subsequently, AD 20 nmol was injected to study neuronal activity indicated by the presence of Fos protein and its co-localization with serotonergic neurons in the MRN and DRN of naive rats with or without access to food during the recording of behavior. The administration of AD 20 nmol increased Fos expression and double labeling with serotonergic neurons in the DRN in rats with access to food, but not in animals without access. No statistically significant changes in Fos expression were observed in the MRN in any of the experimental conditions tested. These results suggest that DRN serotonergic and non-serotonergic neurons are activated by post-prandial signals. In contrast, the absence of Fos expression in the MRN suggests that this nucleus does not participate in the circuit involved in the control of post-prandial satiety.