Number Of c-Fos Positive Cells Research Articles

Involvement of descending pain control system regulated by orexin receptor signaling in the induction of central post-stroke pain in mice

Central post-stroke pain (CPSP) is a type of neuropathic pain for which the mechanism and relevant drug pathways remain unknown. Recently, it was reported that intracerebroventricular (ICV) administration of orexin-A suppresses pain and ischemia. In this study, we tested the role of orexin-A in CPSP induction in mice. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). CPSP was assessed by von Frey test. Colocalization of orexin 1 receptor (OX1R) with various neuron markers were determined by double-immunofluorescence. The hindpaw withdrawal responses to mechanical stimuli were significantly increased 3 days post-BCAO compared with those of sham groups. ICV injection of orexin-A dose-dependently suppressed BCAO-induced mechanical allodynia. These effects were inhibited by pre-treatment with SB334867 (an OX1R antagonist; ICV injection), yohimbine (a noradrenaline α2 receptor antagonist; intrathecal (IT) injection), and WAY100635 (a serotonin 5-HT1A receptor antagonist; IT injection), but not TCS OX2 29 (an OX2R antagonist; ICV injection). OX1R colocalized with TH (a noradrenergic neuron marker) and TPH (a serotonergic neuron marker) in the locus ceruleus (LC) and the rostral ventromedial medulla (RVM), respectively. The number of c-Fos positive cells in the LC and the RVM of BCAO mice was increased at 90 min after ICV injection of orexin-A compared to saline group. These results indicate that orexin-A/OX1R signaling plays an important role through activation of the descending pain control system in the induction of CPSP in mice.

GPR40 full agonism exerts feeding suppression and weight loss through afferent vagal nerve.

GPR40/FFAR1 is a Gq protein-coupled receptor expressed in pancreatic β cells and enteroendocrine cells, and mediates insulin and incretin secretion to regulate feeding behavior. Several GPR40 full agonists have been reported to reduce food intake in rodents by regulating gut hormone secretion in addition to their potent glucose-lowering effects; however, detailed mechanisms of feeding suppression are still unknown. In the present study, we characterized T-3601386, a novel compound with potent full agonistic activity for GPR40, by using in vitro Ca2+ mobilization assay in Chinese hamster ovary (CHO) cells expressing FFAR1 and in vivo hormone secretion assay. We also evaluated feeding suppression and weight loss after the administration of T-3601386 and investigated the involvement of the vagal nerve in these effects. T-3601386, but not a partial agonist fasiglifam, increased intracellular Ca2+ levels in CHO cells with low FFAR1 expression, and single dosing of T-3601386 in diet-induced obese (DIO) rats elevated plasma incretin levels, suggesting full agonistic properties of T-3601386 against GPR40. Multiple doses of T-3601386, but not fasiglifam, in DIO rats showed dose-dependent weight loss accompanied by feeding suppression and durable glucagon-like peptide-1 elevation, all of which were completely abolished in Ffar1-/- mice. Immunohistochemical analysis in the nuclei of the solitary tract demonstrated that T-3601386 increased the number of c-Fos positive cells, which also disappeared in Ffar1-/- mice. Surgical vagotomy and drug-induced deafferentation counteracted the feeding suppression and weight loss induced by the administration of T-3601386. These results suggest that T-3601386 exerts incretin release and weight loss in a GPR40-dependent manner, and that afferent vagal nerves are important for the feeding suppression induced by GPR40 full agonism. Our novel findings raise the possibility that GPR40 full agonist can induce periphery-derived weight reduction, which may provide benefits such as less adverse effects in central nervous system compared to centrally-acting anti-obesity drugs.

Experimental study of the effect of the sciatic nerve elongation on pain in rats

To investigate the effect of the sciatic nerve elongation on pain in rats. Thirty-six adult male Wistar rats of SPF grade, weighing 250-300 g. Eighteen of them were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A), nerve no-elongation group (group B), and nerve ligation group (group C). The model of 10-mm sciatic nerve defect was established in all 3 groups. The sciatic nerve was extended at a speed of 1 mm/d for 14 days in group A. The group B was only installed with external fixation. The nerve stumps were ligated in the group C. At 3, 7, 10, and 14 days after operation, the foot injury was evaluated by the autotomy scoring scale. At 14 days after operation, the dorsal root ganglia (DRG) of L 4-S 1 spinal cord of rats in each group was observed by tumor necrosis factor α (TNF-α) immunohistochemical staining, and the primary antibodies were replaced by pure serum as negative control group. Another 18 rats were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A1), nerve no-elongation group (group B1), positive control group (group C1). In groups A1 and B1, the 10-mm long sciatic nerve defect model was established by the same method as groups A and B, and then fixed with external fixation. Nerve elongation was done or not done without anesthesia at 3 days after operation. In group C1, no modeling was done and 20 μL 2.5% formaldehyde was injected into the toes. After 90 minutes, the dorsal horn of spinal cord of L 4-S 1 segment of rats was cutting for c-Fos immunohistochemical staining and the number of positive cells was counted. Primary antibodies were replaced with pure serum as negative control group. The autotomy scores of rats in groups B and C gradually increased postoperatively, and group A remained stable at 0.25±0.50. The scores of group C were significantly higher than those of group A and group B at each time point postoperatively ( P<0.05). The scores of group A were significantly lower than those of group B at 10 and 14 days postoperatively ( P<0.05). TNF-α immunohistochemical staining showed that the TNF-α expression in group A was weak, slightly positive (+/-); in group B was positive (+); in group C was strongly positive (++); and the negative control group had no TNF-α expression (-). c-Fos immunohistochemical staining showed that the c-Fos expressions in groups A1 and B1 were weak positive, in group C1 was strong positive, and negative control group had no c-Fos positive expression. The number of c-Fos positive cells in groups A1, B1, C1, and negative control group were (21.5±6.6), (19.3±8.1), (95.6±7.4), and 0 cells/field, respectively, and group C1 was significantly higher than groups A1 and B1 ( P<0.05), there was no significant difference between group A1 and group B1 ( P>0.05). Nerve elongation does not cause obvious pain neither during the operation of elongation nor throughout the whole elongation.

The effect of herpes simplex virus vector-mediated gene therapy of protein phosphatase 1α on bladder overactivity and nociception.

We studied the effect of herpes simplex virus (HSV) vectors-based gene transfer of protein phosphatase 1α (PP1α) on bladder hypersensitivity in rats. Using adult female Sprague-Dawley rats, non-replicating HSV vectors carrying PP1α or green fluorescent protein (GFP) were injected into the bladder wall. At one week after vector inoculation, cystometry and Western blot assay were performed, whereas the other experiments were performed at 2 weeks after vector inoculation. GFP-expressing cells were identified in the bladder as well as in L6/S1 dorsal root ganglia at 14 days. In cystometry, intercontraction intervals (ICI) after resiniferatoxin (RTx; TRPV1 agonist) irrigation was significantly reduced in the PP1α group in comparison with the GFP group. Moreover, RTx-induced freezing behavior events were observed significantly more frequently in the PP1α group than the GFP group. The number of c-Fos positive cells in the L6 spinal dorsal horn was significantly less in the PP1α group than in the GFP group. Western blot assay revealed lower levels of phosphorylated inositol 1, 4, 5-triphosphate receptor (p-IP3 R), and phosphorylated TRPV1 in the PP1α compared with the GFP group. HSV vectors-mediated PP1α gene therapy may be an alternative treatment modality for cystitis-related hypersensitive bladder condition at least in part via modulation of the IP3 R signaling pathway.

Effect of dexmedetomidine on visceral pain in rats: the role of α2 adrenergic receptors in locus coeruleus

Objective To evaluate the effect of dexmedetomidine on visceral pain in rats and the role of α2 adrenergic receptors in locus coeruleus (LC). Methods Thirty-two healthy adult male Sprague-Dawley rats, weighing 250-300 g, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), visceral pain group (group VP), dexmedetomidine group (group DEX) and α2-adrenergic receptor antagonist atipamezole group (group AP). α2-adrenergic receptor antagonist atipamezole 522 μg/kg was intramuscularly injected in group AP, and the equal volume of normal saline was given instead in C, VP and DEX groups.At 10 min after intramuscular injection, dexmedetomidine 10 μg/kg was injected via the tail vein in DEX and AP groups, and the equal volume of normal saline was given instead in C and VP groups.VP, DEX and AP groups received intraperitoneal injection of 0.9% acetic acid 10 ml/kg to make the visceral pain model at 15 min after injection via the tail vein.The cumulative visceral pain score was recorded within 60 min after acetic acid injection.The number of c-fos positive cells in LC was detected by immunohistochemistry, and the content of norepinephrine (NA) in the spinal cord were detected by enzyme-linked immunosorbent assay at 2 h after acetic acid injection. Results Compared with group C, the cumulative visceral pain scores, the number of c-fos positive cells in LC and content of NA in the spinal cord were significantly increased in VP, DEX and AP groups (P<0.05). Compared with group VP, the cumulative visceral pain scores, the number of c-fos positive cells in LC and content of NA in the spinal cord were significantly decreased in DEX and AP groups (P<0.05). Compared with group DEX, the cumulative visceral pain scores, the number of c-fos positive cells in LC and content of NA in the spinal cord were significantly increased in group AP (P<0.05). Conclusion Dexmedetomidine can alleviate visceral pain in rats, and the mechanism is partially related to activating α2 adrenergic receptors in LC. Key words: Dexmedetomidine; Visceral; Pain; Locus coeruleus; Receptors, adrenergic alpha-2

Activation of Dorsomedial Hypothalamic Neurons Promotes Physical Activity and Decreases Food Intake and Body Weight in Zucker Fatty Rats.

Previous reports have shown that running wheel activity or voluntary exercise prevents hyperphagia and obesity in various animal models of obesity, but such effects seem only minimal in obese animals lacking leptin or leptin receptors. The mechanisms underlying this ineffectiveness remain unclear. Here, we identified the action of neuronal activation in the dorsomedial hypothalamus (DMH) in modulating physical activity, food intake and body weight using leptin receptor mutant obese Zucker (Lepr(fa), ZF) and Koletsky (Lepr(fak), SHROB) rats. Ad lib-fed SHROB rats with locked running wheels became hyperphagic and gained body weight rapidly. These alterations were not ameliorated in ad lib-fed SHROB rats with voluntary access to running wheels, but the body weight of SHROB rats with running wheel access was significantly decreased when they were pair-fed to the amounts consumed by lean controls. Determinations of hypothalamic gene expression revealed that sedentary ad lib-fed SHROB rats had increased expression of neuropeptide Y (Npy) and decreased expression of pro-opiomelanocortin (Pomc) in the arcuate nucleus (ARC). Both ARC Npy and Pomc expression were further altered under running and pair-fed conditions, indicating that both genes are appropriately regulated in response to increased energy demands or alterations caused by running activity and food restriction. Strikingly, c-Fos immunohistochemistry revealed that while voluntary running activity elevated the number of c-Fos positive cells in the DMH (particularly in the ventral and caudal subregions) of intact rats, such activation was not observed in ZF rats. Using adeno-associated virus (AAV)-mediated expression of the designer receptors hM3D(Gq) in the ventral and caudal DMH of ZF rats, we found that chemogenetic stimulation of neurons in these DMH subregions via injection of the designer drug clozapine N-oxide (CNO) significantly increased their running activity and reduced their food intake and body weight. Together, these results demonstrate that activation of ventral and caudal DMH neurons promotes physical activity and decreases food intake and body weight and suggest that intact DMH neural signaling is likely crucial for exercise-induced reductions of food intake and body weight in obese rats lacking leptin receptors.

Angiotensin‐(1‐7)‐Mediated Increases in Energy Expenditure Appear Independent of the Hypothalamic Melanocortin System

Obesity is a major public health problem that greatly increases risk for cardiovascular disease and type II diabetes. Obesity represents an energy imbalance where the amount of calories consumed exceeds the amount of calories expended. This energy balance is in part controlled by activation of proopiomelanocortin (POMC) neurons or inhibition of agouti‐related protein (AgRP) neurons within the arcuate nucleus (ARC) of the hypothalamus, which activates downstream melanocortin pathways to produce satiety and increase energy expenditure. Our preliminary data show that the beneficial renin‐angiotensin system hormone angiotensin (Ang)‐(1‐7) may be an important contributor to energy balance, as it attenuates high fat diet (HFD)‐induced obesity in mice by browning subcutaneous white fat to enhance energy expenditure. In this study, we tested the hypothesis that the beneficial effects of Ang‐(1‐7) on energy expenditure may be due to actions on POMC or AgRP neurons in the ARC. To test this hypothesis, we first examined the ability of an acute subcutaneous injection of Ang‐(1‐7) to induce neuronal activation in the ARC, as measured by c‐fos gene expression, in chow fed mice. We found that Ang‐(1‐7) significantly increased the number of c‐fos positive cells within the ARC compared with saline (43±5 Ang‐(1‐7) vs. 19±3 saline; p=0.022; n=3 per group). We then performed chronic experiments in which eight‐week old male C57BL/6J mice were implanted with subcutaneous osmotic mini‐pumps to deliver saline or Ang‐(1‐7) [400 ng/kg/min]. Immediately following mini‐pump implantation, mice were placed on a 60% high fat diet (HFD) or matched control chow diet for 12 weeks. The groups of mice included in this chronic study were: (1) chow diet with saline infusion (n=5); (2) HFD with saline infusion (n=6); and (3) HFD with Ang‐(1‐7) infusion [n=9]. At the end of treatment, the hypothalamus from each mouse was collected to assess for gene expression of mas receptor, POMC, and AgRP using quantitative real‐time polymerase chain reaction methods. We found that HFD reduced gene expression for POMC (1.1±0.3 chow vs. 0.4±0.1 HFD; p=0.005) in the hypothalamus, with no significant effect on mas receptor (1.2±0.3 chow vs. 0.7±0.1 HFD; p=0.134) or AgRP (1.3±0.5 chow vs. 1.1±0.2 HFD; p=0.856). There was no effect of Ang‐(1‐7) on POMC (0.4±0.1; p=0.998), mas receptor (0.7±0.1; p=0.995) or AgRP (1.0±0.1; p=0.996) gene expression in HFD mice. These findings provide evidence that Ang‐(1‐7) can acutely induce activation of neurons in the ARC. Chronic Ang‐(1‐7) treatment was not able to restore obesity‐induced reductions in POMC gene expression, however, despite enhancing energy expenditure. Further studies are needed to evaluate if beneficial effects of Ang‐(1‐7) on energy balance involve changes in POMC neuronal activity, or melanocortin‐independent pathways.Support or Funding InformationThis study was supported by NIH grant HL122507.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P 77 Optogenetic stimulations of striatal cholinergic interneurons in an animal model of dystonia

Dystonias are movement disorders, defined by sustained or intermittent muscle contractions causing twisting movements and postures. The most prevalent inherited form of dystonia is caused by a mutation in the gene for torsin A (DYT1, ΔGAG) with incomplete penetrance. It has been hypothesized that an increased activity of cholinergic interneurons in the striatum, resulting in abnormal synaptic plasticity, plays an important role in the disease pathophysiology. However, this hypothesis is merely based on ex vivo electrophysiological recordings in brain slices of animal models which do not show a dystonic phenotype. Here we aim to substantiate the role of this alteration for motor dysfunctions in vivo by using optogenetic activation of striatal cholinergic interneurons in freely behaving mice. For this purpose we crossed Dyt1 ΔGAG heterozygous knock-in mice (DYT1) with mice expressing a light-dependent cation channel (Channelrhodopsin2, ChR2), essential for optogenetic examinations, specifically in cholinergic interneurons (Chat promotor). ChR2 is opened by blue light (wavelength 470 nm), leading to depolarization and acetylcholine release. The light (LED) is conveyed by optical cannulae, which were chronically implanted by stereotaxic surgery into the striatum of DYT1/ChR2 mice and wildtype (WT)/ChR2 littermates. First, we ensured that ChR2 is expressed in cholinergic interneurons of ChR2 + mice by immunohistochemistry and confocal microscopy and that these neurons respond to blue light exposure in brain slices. Subsequently, effects of different stimulation parameters (e.g. pulse width, Hz) on motor activity were examined in WT/ChR2 ( n = 9) and DYT1/ChR2 mice ( n = 6). Bilateral and unilateral photostimulations with blue LED light were compared to yellow LED light which does not open ChR2 (negative control). As expected, yellow light had no influence on behavior. Effects of bilateral stimulations with blue light were more robust than unilateral stimulations. Selected stimulation parameters induced significant increases of general activity (increased distance moved) specifically in DYT1/ChR2 mice compared to before or after stimulation, while there were no effects in WT/ChR2. This substantiates a specific role of striatal cholinergic neurons in motor activity in DYT1 dystonia. We have recently shown that DYT1 KI mice, which do not develop overt dystonia, present with sensorimotor deficits in specific behavioral paradigms. Optogenetic activation of cholinergic interneurons ameliorated this deficit temporarily. Preliminary data of an investigation of neuronal activity in the striatum after stimulation indicated an increased number of ChR2 cells positive for c-Fos in Dyt1/ChR2 ( n = 4) in comparison to WT/ChR2 ( n = 5) while there was no difference in total number of c-Fos positive cells. Total number of c-Fos positive cells did also not differ between naive WT/ChR2 and Dyt1/ChR2 mice, supporting that increased c-Fos expression in cholinergic interneurons is the expected effect of optogenetic stimulation on neuronal activity. We now perform comprehensive studies with short- and long-term stimulations in the Dyt1 mice to clarify the importance of overactive striatal cholinergic interneurons for the manifestation and abnormal striatal plasticity in DYT1 dystonia. For this purpose optogenetics will be combined with pharmacological, electrophysiological, neurochemical and molecular examinations.

Common Hepatic Branch of Vagus Nerve-Dependent Expression of Immediate Early Genes in the Mouse Brain by Intraportal L-Arginine: Comparison with Cholecystokinin-8.

Information from the peripheral organs is thought to be transmitted to the brain by humoral factors and neurons such as afferent vagal or spinal nerves. The common hepatic branch of the vagus (CHBV) is one of the main vagus nerve branches, and consists of heterogeneous neuronal fibers that innervate multiple peripheral organs such as the bile duct, portal vein, paraganglia, and gastroduodenal tract. Although, previous studies suggested that the CHBV has a pivotal role in transmitting information on the status of the liver to the brain, the details of its central projections remain unknown. The purpose of the present study was to investigate the brain regions activated by the CHBV. For this purpose, we injected L-arginine or anorexia-associated peptide cholecystokinin-8 (CCK), which are known to increase CHBV electrical activity, into the portal vein of transgenic Arc-dVenus mice expressing the fluorescent protein Venus under control of the activity-regulated cytoskeleton-associated protein (Arc) promotor. The brain slices were prepared from these mice and the number of Venus positive cells in the slices was counted. After that, c-Fos expression in these slices was analyzed by immunohistochemistry using the avidin-biotin-peroxidase complex method. Intraportal administration of L-arginine increased the number of Venus positive or c-Fos positive cells in the insular cortex. This action of L-arginine was not observed in CHBV-vagotomized Arc-dVenus mice. In contrast, intraportal administration of CCK did not increase the number of c-Fos positive or Venus positive cells in the insular cortex. Intraportal CCK induced c-Fos expression in the dorsomedial hypothalamus, while intraportal L-arginine did not. This action of CCK was abolished by CHBV vagotomy. Intraportal L-arginine reduced, while intraportal CCK increased, the number of c-Fos positive cells in the nucleus tractus solitarii in a CHBV-dependent manner. The present results suggest that the CHBV can activate different brain regions depending on the nature of the peripheral stimulus.

In vitro Magnetic Stimulation: A Simple Stimulation Device to Deliver Defined Low Intensity Electromagnetic Fields.

Non-invasive brain stimulation (NIBS) by electromagnetic fields appears to benefit human neurological and psychiatric conditions, although the optimal stimulation parameters and underlying mechanisms remain unclear. Although, in vitro studies have begun to elucidate cellular mechanisms, stimulation is delivered by a range of coils (from commercially available human stimulation coils to laboratory-built circuits) so that the electromagnetic fields induced within the tissue to produce the reported effects are ill-defined. Here, we develop a simple in vitro stimulation device with plug-and-play features that allow delivery of a range of stimulation parameters. We chose to test low intensity repetitive magnetic stimulation (LI-rMS) delivered at three frequencies to hindbrain explant cultures containing the olivocerebellar pathway. We used computational modeling to define the parameters of a stimulation circuit and coil that deliver a unidirectional homogeneous magnetic field of known intensity and direction, and therefore a predictable electric field, to the target. We built the coil to be compatible with culture requirements: stimulation within an incubator; a flat surface allowing consistent position and magnetic field direction; location outside the culture plate to maintain sterility and no heating or vibration. Measurements at the explant confirmed the induced magnetic field was homogenous and matched the simulation results. To validate our system we investigated biological effects following LI-rMS at 1 Hz, 10 Hz and biomimetic high frequency, which we have previously shown induces neural circuit reorganization. We found that gene expression was modified by LI-rMS in a frequency-related manner. Four hours after a single 10-min stimulation session, the number of c-fos positive cells increased, indicating that our stimulation activated the tissue. Also, after 14 days of LI-rMS, the expression of genes normally present in the tissue was differentially modified according to the stimulation delivered. Thus we describe a simple magnetic stimulation device that delivers defined stimulation parameters to different neural systems in vitro. Such devices are essential to further understanding of the fundamental effects of magnetic stimulation on biological tissue and optimize therapeutic application of human NIBS.

Distinct contributions of reactive oxygen species in amygdala to bee venom-induced spontaneous pain-related behaviors

Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, play essential roles in physiological plasticity and are also involved in the pathogenesis of persistent pain. Roles of peripheral and spinal ROS in pain have been well established, but much less is known about ROS in the amygdala, a brain region that plays an important role in pain modulation. The present study explored the contribution of ROS in the amygdala to bee venom (BV)-induced pain behaviors. Our data show that the amygdala is activated following subcutaneous BV injection into the left hindpaw, which is reflected in the increased number of c-Fos positive cells in the central and basolateral amygdala nuclei in the right hemisphere. Stereotaxic administration of a ROS scavenger (tempol, 10mM), NADPH oxidase inhibitor (baicalein, 5mM) or lipoxygenase inhibitor (apocynin, 10mM) into the right amygdala attenuated the BV-induced spontaneous licking and lifting behaviors, but had no effect on BV-induced paw flinch reflexes. Our study provides further evidence for the involvement of the amygdala in nociceptive processing and pain behaviors, and that ROS in amygdala may be a potential target for treatment strategies to inhibit pain.

Transcutaneous Cervical Vagus Nerve Stimulation Ameliorates Acute Ischemic Injury in Rats.

Direct stimulation of the vagus nerve in the neck via surgically implanted electrodes is protective in animal models of stroke. We sought to determine the safety and efficacy of a non-invasive cervical VNS (nVNS) method using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery occlusion (MCAO). nVNS was initiated variable times after MCAO in rats (n = 33). Control animals received sham stimulation (n = 33). Infarct volume and functional outcome were assessed on day 7. Brains were processed by immunohistochemistry for microglial activation and cytokine levels. The ability of nVNS to activate the nucleus tractus solitarius (NTS) was assessed using c-Fos immunohistochemistry. Infarct volume was 43.15 ± 3.36 percent of the contralateral hemisphere (PCH) in control and 28.75 ± 4.22 PCH in nVNS-treated animals (p < 0.05). The effect of nVNS on infarct size was consistent when stimulation was initiated up to 4 hours after MCAO. There was no difference in heart rate and blood pressure between control and nVNS-treated animals. The number of c-Fos positive cells was 32.4 ± 10.6 and 6.2 ± 6.3 in the ipsilateral NTS (p < 0.05) and 30.4 ± 11.2 and 5.8 ± 4.3 in the contralateral NTS (p < 0.05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF-α positive cells and increased the number of HMGB1 positive cells. nVNS inhibits ischemia-induced immune activation and reduces the extent of tissue injury and functional deficit in rats without causing cardiac or hemodynamic adverse effects when initiated up to 4 hours after MCAO.

Effects of aripiprazole on caffeine-induced hyperlocomotion and neural activation in the striatum.

Aripiprazole is an antipsychotic that acts as a partial agonist at dopamine D2 receptors. In addition to its antipsychotic activity, this compound blocks the effects of some psychostimulant drugs. It has not been verified, however, if aripiprazole interferes with the effects of caffeine. Hence, this study tested the hypothesis that aripiprazole prevents caffeine-induced hyperlocomotion and investigated the effects of these drugs on neural activity in the striatum. Male Swiss mice received injections of vehicle or antipsychotic drugs followed by vehicle or caffeine. Locomotion was analyzed in a circular arena and c-Fos protein expression was quantified in the dorsolateral, dorsomedial, and ventrolateral striatum, and in the core and shell regions of nucleus accumbens. Aripiprazole (0.1, 1, and 10 mg/kg) prevented caffeine (10 mg/kg)-induced hyperlocomotion at doses that do not change basal locomotion. Haloperidol (0.01, 0.03, and 0.1 mg/kg) also decreased caffeine-induced hyperlocomotion at all doses, although at the two higher doses, this compound reduced basal locomotion. Immunohistochemistry analysis showed that aripiprazole increases c-Fos protein expression in all regions studied, whereas caffeine did not alter c-Fos protein expression. Combined treatment of aripiprazole and caffeine resulted in a decrease in the number of c-Fos positive cells as compared to the group receiving aripiprazole alone. In conclusion, aripiprazole prevents caffeine-induced hyperlocomotion and increases neural activation in the striatum. This latter effect is reduced by subsequent administration of caffeine. These results advance our understanding on the pharmacological profile of aripiprazole.

TRPV4 activation at the physiological temperature is a critical determinant of neuronal excitability and behavior.

For homeothermic animals, constant body temperature is an important determinant of brain function. It is well established that changes in brain temperature dynamically influence hippocampal activity. We previously reported that the thermosensor TRPV4 (activated above 34°C) is activated at the physiological temperature in hippocampal neurons and controls neuronal excitability in vitro. Here, we examined if TRPV4 regulates neuronal excitability through its activation at the physiological temperature in vivo. We found that TRPV4-deficient (TRPV4KO) mice exhibit reduced depression-like and social behaviors compared to wild-type (WT) mice, and the number of c-fos positive cells in the dentate gyrus was significantly reduced upon the depression-like behaviors. We measured resting membrane potentials (RMPs) in the hippocampal granule cells from slice preparations at 35°C and found that TRPV4-positive neurons significantly depolarized the RMPs through TRPV4 activation at the physiological temperature. The depolarization increased the spike numbers depending on the enhancement of TRPV4 activation. We also found that theta-frequency electroencephalogram (EEG) activities in TRPV4KO mice during wake periods were significantly reduced compared with those in WT mice. Taken together, we report for the first time that TRPV4 activation at the physiological temperature is important to regulate neuronal excitability and behaviors in mammals.

PD20-05 HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GENE DELIVERY OF PROTEIN PHOSPHATASE 1α REDUCES BLADDER OVERACTIVITY AND

INTRODUCTION AND OBJECTIVES: Increased afferent excitability has been proposed as an important pathophysiological basis of overactive bladder (OAB) and hypersensitive bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Previous studies reported that transient receptor potential vanilloid-1 (TRPV1) receptors greatly contribute to afferent sensitization. We recently found Protein Phosphatase 1a (PP1a) as a modulator of TRPV1 receptor activity using an HSV vector cDNA library screen method to identify genes that antagonize TRPV1 activation. HSV vector-mediated PP1a expression significantly reduced capsaicin-induced thermal hyperalgesia. Therefore, we investigated the effect of HSV vectors-mediated gene delivery of PP1a on bladder overactivity and pain-related behavior in rats. METHODS: Replication-deficient HSV vectors encoding PP1a or green fluorescent protein (GFP) as control were injected into the bladder wall of adult female Sprague-Dawley rats. Cystometry (CMG) under urethane anesthesia was performed 1 week after viral injection to evaluate bladder overactivity induced by resiniferatoxin (RTX, a TRPV1 agonist). RTX-induced nociceptive behavior such as licking (lower abdominal licking) and freezing (motionless headturning) was observed 2 weeks after viral innoculation. GFP expression in the L6/S1 DRG and the bladder as well as c-Fos positive cells in the L6 spinal cord dorsal horn were evaluated. The expression of p-ERK, p-Inositol trisphosphate receptor (p-IP3R), and p-TRPV1 in the bladder was estimated by Western blot. RESULTS: GFP expression was seen in L6/S1 DRG as well as in the bladder. In CMG, the PP1a group showed a significantly (p1⁄40.03) smaller reduction (46 1%) in intercontraction intervals after RTX infusion than the GFP group (64 2%). The number of RTX-induced freezing behavior, which is correlated with bladder pain sensation, was significantly (p<0.001) decreased in the PP1a vs. GFP groups. The number of c-Fos positive cells in the L6 spinal dorsal horn was significantly (p<0.01) smaller in the PP1a vs. the GFP group (33 4 vs. 59 6 cells per section). Western blot revealed that p-ERK, p-IP3R, and p-TRPV1 was less expressed in the PP1a vs. the GFP group (p1⁄40.07, 0.01, and 0.04, respectively) CONCLUSIONS: These results indicate that HSV vectorsmediated gene delivery of PP1a can suppress TRPV1-mediated bladder overactivityandpainbehavior, possibly due todecreasedphosphorylation of TRPV1 channels. Thus, PP1a gene thetapy could be a novel treatment of OAB and/or hypersensitive bladder disorders such as IC/BPS.

Tissue plasminogen activator modulates emotion in a social context

Tissue plasminogen activator (tPA) is known to play physiologically and pathologically crucial roles in the central nervous system. However, it is still obscure whether it affects social behavior. We investigated social behavior and neuronal activation after social stimulation in tPA knockout (KO) mice. In a resident-intruder test, the latency to the first contact was significantly delayed in tPA KO mice compared with that in tPA heterogenic (Het) mice. However, tPA KO mice spent significantly more time undertaking active behavior than tPA Het mice did. In a sociability test, tPA KO mice significantly spent more time and walked a greater distance in the chamber containing an empty cage than tPA Het mice. Furthermore, tPA KO mice approached an empty cage more frequently than tPA Het mice did. In a social novelty test, there was no difference in the duration spent sniffing a stimulator mouse between genotypes. However, tPA KO mice approached even a familiar mouse more frequently than tPA Het mice did. tPA KO mice spent similar durations in a chamber containing a familiar mouse and that containing an unfamiliar mouse, and tPA KO mice walked a significantly greater distance in the former chamber than tPA Het mice did. Furthermore, at the cingulate and prelimbic cortices, the number of cFos-positive cells was significantly increased in tPA KO mice compared with that in tPA Het mice after social stimulation. Our results suggest that tPA modulates emotion in a social context through the function of the prefrontal cortex.

MP1-03 HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GENE DELIVERY OF PROTEIN PHOSPHATASE 1α REDUCES BLADDER OVERACTIVITY AND NOCICEPTION IN RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 2014MP1-03 HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GENE DELIVERY OF PROTEIN PHOSPHATASE 1α REDUCES BLADDER OVERACTIVITY AND NOCICEPTION IN RATS Tsuyoshi Majima, Naoki Kawamorita, Hiroki Okada, Yasuhito Funahashi, Momokazu Gotoh, William Goins, Joseph Glorioso, Justus Cohen, Bonnie Hall, Asaff Harel, and Naoki Yoshimura Tsuyoshi MajimaTsuyoshi Majima More articles by this author , Naoki KawamoritaNaoki Kawamorita More articles by this author , Hiroki OkadaHiroki Okada More articles by this author , Yasuhito FunahashiYasuhito Funahashi More articles by this author , Momokazu GotohMomokazu Gotoh More articles by this author , William GoinsWilliam Goins More articles by this author , Joseph GloriosoJoseph Glorioso More articles by this author , Justus CohenJustus Cohen More articles by this author , Bonnie HallBonnie Hall More articles by this author , Asaff HarelAsaff Harel More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.101AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Increased afferent excitability has been proposed as an important pathophysiological basis of overactive bladder (OAB) and hypersensitive bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). It has also been reported that transient receptor potential vanilloid-1 (TRPV1) receptors predominantly expressed in C-fiber afferent pathways greatly contribute to afferent sensitization in these disease conditions. We recently found Protein Phosphatase 1α (PP1α) as a modulator of TRPV1 receptor activity using an HSV vector cDNA library screen method to identify genes that inhibit TRPV1 activation, and showed that HSV vector-mediated PP1α expression led to the reduction in capsaicin-induced thermal hyperalgesia. Therefore, this led us to investigate the effect of HSV vectors-mediated gene delivery of PP1α on TRPV1-mediated bladder overactivity and pain-related behavior in rats. METHODS Replication-deficient HSV vectors encoding PP1α or green fluorescent protein (GFP) as control were injected into the bladder wall of adult female Sprague-Dawley rats. Cystometry (CMG) under urethane anesthesia was performed 1 week after viral injection to evaluate bladder overactivity induced by resiniferatoxin (RTX, a TRPV1 agonist). RTX-induced nociceptive behavior such as licking (lower abdominal licking) and freezing (motionless head-turning) was observed 2 weeks after viral injection. Using immunohistochemistry, GFP expression in L6/S1 DRG and the bladder as well as c-Fos positive cells in the L6 spinal cord dorsal horn were evaluated. RESULTS GFP expression was seen in L6/S1 DRG sections. In CMG, the PP1α group showed a significantly (p<0.05) smaller reduction (46.5±1.7%) in intercontraction intervals after RTX infusion than the GFP group (65.7±3.8%). The number of RTX-induced freezing behavior, which is correlated with bladder pain sensation, was significantly (p<0.001) decreased in PP1α vs. GFP groups. The number of c-Fos positive cells in the L6 spinal dorsal horn was significantly (p<0.01) smaller in PP1α vs. GFP rats (24±4 vs. 59±6 cells per section). CONCLUSIONS These results indicate that HSV vectors injected into the bladder wall are transported to lumbosacral DRGs that contain bladder afferent neurons, and that PP1α gene delivery in the bladder is effective in suppressing TRPV1-mediated bladder overactivity and pain behavior. Thus, HSV-mediated PP1α gene therapy could be effective for the treatment of OAB and/or hypersensitive bladder disorders such as IC/BPS. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e1-e2 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Tsuyoshi Majima More articles by this author Naoki Kawamorita More articles by this author Hiroki Okada More articles by this author Yasuhito Funahashi More articles by this author Momokazu Gotoh More articles by this author William Goins More articles by this author Joseph Glorioso More articles by this author Justus Cohen More articles by this author Bonnie Hall More articles by this author Asaff Harel More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Spatial memory extinction: A c-Fos protein mapping study

While the neuronal basis of spatial memory consolidation has been thoroughly studied, the substrates mediating the process of extinction remain largely unknown. This study aimed to evaluate the functional contribution of selected brain regions during the extinction of a previously acquired spatial memory task in the Morris water maze. For that purpose, we used adult male Wistar rats trained in a spatial reference memory task. Learning-related changes in c-Fos inmunoreactive cells after training were evaluated in cortical and subcortical regions. Results show that removal of the hidden platform in the water maze induced extinction of the previously reinforced escape behavior after 16 trials, without spontaneous recovery 24h later. Extinction was related with significantly higher numbers of c-Fos positive nuclei in amygdala nuclei and prefrontal cortex. On the other hand, the lateral mammillary bodies showed higher number of c-Fos positive cells than the control group. Therefore, in contrast with the results obtained in studies of classical conditioning, we show the involvement of diencephalic structures mediating this kind of learning. In summary, our findings suggest that medial prefrontal cortex, the amygdala complex and diencephalic structures like the lateral mammillary nuclei are relevant for the extinction of spatial memory.

Influence of chronic moderate sleep restriction and exercise training on anxiety, spatial memory, and associated neurobiological measures in mice

Sleep deprivation can have deleterious effects on cognitive function and mental health. Moderate exercise training has myriad beneficial effects on cognition and mental health. However, physiological and behavioral effects of chronic moderate sleep restriction and its interaction with common activities, such as moderate exercise training, have received little investigation. The aims of this study were to examine the effects of chronic moderate sleep restriction and moderate exercise training on anxiety-related behavior, spatial memory, and neurobiological correlates in mice. Male mice were randomized to one of four 11-week treatments in a 2 [sleep restriction (∼4h loss/day) vs. ad libitum sleep] × 2 [exercise (1h/day/6 d/wk) vs. sedentary activity] experimental design. Anxiety-related behavior was assessed with the elevated-plus maze, and spatial learning and memory were assessed with the Morris water maze. Chronic moderate sleep restriction did not alter anxiety-related behavior, but exercise training significantly attenuated anxiety-related behavior. Spatial learning and recall, hippocampal cell activity (i.e., number of c-Fos positive cells), and brain derived neurotrophic factor were significantly lower after chronic moderate sleep restriction, but higher after exercise training. Further, the benefit of exercise training for some memory variables was evident under normal sleep, but not chronic moderate sleep restriction conditions. These data indicate clear detrimental effects of chronic moderate sleep restriction on spatial memory and that the benefits of exercise training were impaired after chronic moderate sleep restriction.

Neonatal whisker clipping alters behavior, neuronal structure and neural activity in adult rats

Early experience plays critical roles during the development of sensory systems. For example, neonatal surgical manipulations of the whiskers in rodents lead to altered neural activity and behaviors later in life. However, while surgical procedures damage the sensory pathway; it is hard to examine the impact of whisker deprivation on adult animals. To address this issue, we performed a neonatal whisker clipping (WC0-3) paradigm, a non-invasive procedure, from the day of birth (P0) to postnatal day (P) 3, and examined behavioral performances in their adult age. With fully regrown whiskers, the WC0-3 rats exhibited shorter crossable distance than controls in a gap-crossing task, suggesting a defect in their whisker-specific tactile function. In their somatosensory cortex, the layer IV spiny stellate neurons had reduced dendritic complexity and spine density. After exploration in a novel environment, the expression of an activity-dependent immediate early gene, c-fos, increased dramatically in the somatosensory cortex. However, in WC0-3 rats, the number of c-Fos positive cells was less than those in control rats, indicating a fault in transducing sensory-related neural activity between cortical layers in WC0-3 rats. Together, our results demonstrate the roles of early tactile experience on the establishment of layer-specific excitatory connection in the barrel cortex. Early sensory insufficiency would leave long-lasting functional deficits in the sensory system.