Ventromedial prefrontal area 14 provides opposing regulation of threat and reward-elicited responses in the common marmoset.

The ventromedial prefrontal cortex (vmPFC) is a key brain structure implicated in mood and anxiety disorders, based primarily on evidence from correlational neuroimaging studies. Composed of a number of brain regions with distinct architecture and connectivity, dissecting its functional heterogeneity will provide key insights into the symptomatology of these disorders. Focusing on area 14, lying on the medial and orbital surfaces of the gyrus rectus, this study addresses a key question of causality. Do changes in area 14 activity induce changes in threat- and reward-elicited responses within the nonhuman primate, the common marmoset, similar to that seen in mood and anxiety disorders? Area 14 overactivation was found to induce heightened responsivity to uncertain, low-imminence threat while blunting cardiovascular and behavioral anticipatory arousal to high-value food reward. Conversely, inactivation enhanced the arousal to high-value reward cues while dampening the acquisition of cardiovascular and behavioral responses to a Pavlovian threat cue. Basal cardiovascular activity, including heart rate variability and sympathovagal balance, which are dysfunctional in mood and anxiety disorders, are insensitive to alterations in area 14 activity as is the extinction of conditioned threat responses. The distinct pattern of dysregulation compared to neighboring region area 25 highlights the heterogeneity of function within vmPFC and reveals how the effects of area 14 overactivation on positive and negative reactivity mirror symptoms of anhedonia and anxiety that are so often comorbid in mood disorders.

The role of Pak-interacting exchange factor-ß phosphorylation at serines 340 and 583 by PKCγ in dopamine release.

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-ß (ßPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates ßPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated ßPIX in vivo. Classical PKC inhibitors and ßPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type ßPIX, and not the ßPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by ßPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of ßPIX at Ser340 and Ser583 has pivotal roles in Ca(2+)-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through ß2PIX phosphorylation. The PKCγ-ßPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

Dual 5-HT2A and 5-HT2C Receptor Inverse Agonist That Affords In Vivo Antipsychotic Efficacy with Minimal hERG Inhibition for the Treatment of Dementia-Related Psychosis.

Psychosis is a distressing symptom commonly occurring in people with dementia. To treat Parkinson's disease psychosis, pimavanserin (1), a 5-HT2A receptor inverse agonist having minimal 5-HT2C receptor affinity and no dopamine D2 receptor affinity, was approved in the United States, but not for dementia-related psychosis due to limited efficacy issues. Herein, we report on the identification of a potent and dual 5-HT2A and 5-HT2C receptor inverse agonist 8 having minimal hERG inhibition, after having demonstrated the involvement of both 5-HT2A and 5-HT2C receptors to deliver antipsychotic efficacy in an MK-801-induced locomotor model and having conducted 5-HT2A and 5-HT2C occupancy studies including a surrogate method. The introduction of a spirocyclopropyl group boosting 5-HT2C affinity in 1 followed by further optimization to control lipophilicity resulted in balanced dual potency and metabolic stability, and mitigating hERG inhibition led to 8 that showed significant antipsychotic efficacy due to the involvement of both receptors.

Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

Donepezil, but not galantamine, blocks muscarinic receptor-mediated in vitro and in vivo responses.

We have found that galantamine, but not donepezil, reversed isolation rearing-induced deficits of prepulse inhibition (PPI) via an activation of muscarinic M1 receptors. To explain this difference, the present study examined the effects of these acetylcholinesterase inhibitors on muscarinic receptor-mediated responses in in vitro and in vivo systems. Ca(2+) -imaging study showed that donepezil, but not galantamine, blocked a muscarinic agonist carbachol-induced increase in intracellular Ca(2+) levels in SH-SY5Y cells. Moreover, a microdialysis study showed that intraperitoneal administration of donepezil, but not galantamine, attenuated a preferential M1 receptor agonist Ndesmethylclozapine-induced increase in dopamine release in mouse cerebral cortex. These results suggest that donepezil, but not galantamine, has an ability to block muscarinic receptor function and imply that the differential effects may be responsible for the difference in the effects on isolation rearing-induced deficits of PPI between these drugs. Synapse, 2011. © 2011 Wiley-Liss, Inc.

Pharmacological aspects of the acetylcholinesterase inhibitor galantamine.

Several lines of evidence suggest that cholinergic deficits may contribute to the pathophysiology of psychiatric disorders as well as Alzheimer's disease. There is growing clinical evidence that galantamine, currently used for the treatment of Alzheimer's disease, may improve cognitive dysfunction and psychiatric illness in schizophrenia, major depression, bipolar disorder, and alcohol abuse. Since galantamine is a rather weak acetylcholinesterase inhibitor, but has additional allosteric potentiating effects at nicotinic receptors, it affects not only cholinergic transmission but also other neurotransmitter systems such as monoamines, glutamate, and γ-aminobutyric acid (GABA) through its allosteric mechanism. It is likely that these effects may result in more beneficial effects. To understand the underlying mechanism for the clinical effectiveness of galantamine, neuropharmacological studies have been performed in animal models of several psychiatric disorders. These studies suggest that not only the nicotinic receptor-modulating properties but also the muscarinic receptor activation contribute to the antipsychotic effect and improvement of cognitive dysfunction by galantamine. This review summaries the current status on the pharmacology of galantamine, focusing on its effect on neurotransmitter release and pharmacological studies in animal models of psychiatric disorders.

Effects of acute and chronic administration of atomoxetine and methylphenidate on extracellular levels of noradrenaline, dopamine and serotonin in the prefrontal cortex and striatum of mice.

Acute administration of atomoxetine and methylphenidate, attention-deficit/hyperactivity disorder (ADHD) drugs, activates catecholaminergic systems in rat brain, but the effects of their chronic administration are not known. This study examined the effects of acute and chronic administration of ADHD drugs on the extracellular levels of noradrenaline (NA), dopamine (DA) and serotonin (5-HT), and the expression of the neuronal activity marker c-Fos in the prefrontal cortex and striatum of mice. Acute ADHD drugs increased NA and DA, but not 5-HT, levels in the prefrontal cortex of mice. Maximal effects of atomoxetine and methylphenidate were observed at 1 mg/kg and 3 mg/kg, respectively. At these doses, both drugs did not affect the spontaneous locomotor activity of mice. Chronic administration of atomoxetine 1 mg/kg and methylphenidate 3 mg/kg for 21 days also increased NA and DA, but not 5-HT, levels in the prefrontal cortex. The increases in NA levels induced by atomoxetine, but not methylphenidate, were reduced by chronic treatment. In contrast, acute and chronic administration of atomoxetine 1 mg/kg and methylphenidate 3 mg/kg did not affect the monoamine levels in the striatum. Acute and chronic atomoxetine 1 mg/kg and methylphenidate 3 mg/kg increased the expression of c-Fos in the prefrontal cortex, but not in the striatum, to a similar extent. These results suggest that acute and chronic administration of the ADHD drugs selectively activate the prefrontal catecholamine systems in mice.

Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors.

Most advanced knee osteoarthritis (OA) patients experience chronic pain resistant to cyclooxygenase (COX) inhibitors. However, the cells and molecules involved in this advanced OA pain remain poorly understood. In this study, we developed a rat model of advanced knee OA by modification of the monoiodoacetate-induced OA pain model and examined involvement of synovial macrophages in advanced OA pain. Cyclooxygenase inhibitors, such as celecoxib and naproxen, and a steroid were ineffective, but an opioid and anti-nerve growth factor (NGF) antibody was effective for pain management in the advanced OA model. Similar to advanced OA patients, histological analysis indicated severe bone marrow damages, synovitis, and cartilage damage and an increase of macrophages with high expression of interleukin-1ß, NGF, nitric oxide synthase (NOS) 1, NOS2, and COX-2 in the knee joint of the advanced OA model. Intravenous injection of clodronate liposomes depleted synovial macrophages, which decreased the level of not only proinflammatory mediator interleukin-1ß but also NGF in the knee joint, leading to pain suppression in the advanced OA model. These data suggest the involvement of synovial macrophages in advanced knee OA pain resistant to COX inhibitors by increasing proinflammatory mediators, and that drugs targeting synovial macrophages might have potent analgesic effects.

Galantamine and donepezil differently affect isolation rearing-induced deficits of prepulse inhibition in mice.

RATIONALE: Previous studies have shown that alterations in acetylcholine (ACh) receptor subtypes might contribute to cognitive impairment observed in schizophrenia and that choline acetyltransferase activity in the parietal cortex is negatively correlated with the severity of such cognitive impairment. However, clinical data suggest that the acetylcholinesterase (AChE) inhibitors galantamine and donepezil have different effects on negative and cognitive symptoms in schizophrenia. Prepulse inhibition (PPI) deficits--sensory information-processing deficits observed in schizophrenia--may be useful models for studying the efficacy of AChE inhibitors as cognitive enhancers. OBJECTIVES: The present study examined the effects of galantamine and donepezil on PPI deficits induced by an environmental factor and drugs. MATERIALS AND METHODS: In the isolation-rearing model, 3-week-old male ddY mice were housed either in groups of five or six per cage or isolated in cages of the same size for more than 6 weeks. In the drug-induced model, apomorphine 1 mg/kg and MK-801 0.2 mg/kg were administered to 9- to 10-week-old male ddY mice. RESULTS: In isolation-reared mice, galantamine attenuated PPI deficits, while donepezil did not. Galantamine and donepezil both attenuated PPI deficits induced by apomorphine, but not by MK-801. The galantamine-induced improvements in PPI deficits were not prevented by the nicotinic ACh receptor antagonists mecamylamine and methyllycaconitine. CONCLUSIONS: These observations suggest that galantamine and donepezil have different effects on the environmentally induced PPI deficits and that these observations may be relevant to the different effects of these drugs observed clinically in schizophrenia.

Anterior cingulate cortex connectivity is associated with suppression of behaviour in a rat model of chronic pain.

A cardinal feature of persistent pain that follows injury is a general suppression of behaviour, in which motivation is inhibited in a way that promotes energy conservation and recuperation. Across species, the anterior cingulate cortex is associated with the motivational aspects of phasic pain, but whether it mediates motivational functions in persistent pain is less clear. Using burrowing behaviour as an marker of non-specific motivated behaviour in rodents, we studied the suppression of burrowing following painful confirmatory factor analysis or control injection into the right knee joint of 30 rats (14 with pain) and examined associated neural connectivity with ultra-high-field resting state functional magnetic resonance imaging. We found that connectivity between anterior cingulate cortex and subcortical structures including hypothalamic/preoptic nuclei and the bed nucleus of the stria terminalis correlated with the reduction in burrowing behaviour observed following the pain manipulation. In summary, the findings implicate anterior cingulate cortex connectivity as a correlate of the motivational aspect of persistent pain in rodents.

Role of postsynaptic serotonin1A receptors in risperidone-induced increase in acetylcholine release in rat prefrontal cortex.

Most atypical antipsychotic drugs increase acetylcholine release in the prefrontal cortex, but the detailed mechanism is still unknown. The present study examined the role of serotonin (5-HT)1A receptors in risperidone-induced increases in acetylcholine release in rat prefrontal cortex. Systemic administration of risperidone at doses of 1 and 2 mg/kg increased acetylcholine release in the prefrontal cortex in a dose-dependent manner. This increase was antagonized by systemic administration of high doses (1 and 3 mg/kg) of N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635), a 5-HT1A receptor antagonist/dopamine D4 receptor agonist, but not by a low dose (0.1 mg/kg) of the antagonist which antagonizes preferentially presynaptic 5-HT1A autoreceptors. Furthermore, local application of WAY100635 into the prefrontal cortex also attenuated risperidone-induced increases in acetylcholine release. WAY100635 alone did not affect acetylcholine release in the prefrontal cortex. On the other hand, local application of risperidone (3 and 10 microM), the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (1 and 10 microM), and the dopamine D4 receptor antagonist 3-(4-(4-iodophenyl)piperazine-1-yl)methyl-1H-pyrrolo[2,3-b]pyridine (1 and 10 microM) into the cortex did not affect acetylcholine release in the prefrontal cortex. These results suggest that risperidone increases acetylcholine release in the prefrontal cortex through a complex mechanism which is enhanced by prefrontal 5-HT1A receptor activation.

Edaravone, a radical scavenger, inhibits mitochondrial permeability transition pore in rat brain.

Edaravone, a radical scavenger, prevents ischemia/reperfusion injury in the brain, but the detailed mechanism is not known. This study examines the effect of edaravone on mitochondrial permeability transition pore (PTP) in rat brain. Edaravone at 10 - 100 microM inhibited Ca(2+)- and H(2)O(2)-induced swelling of mitochondria isolated from rat brain. Addition of Ca(2+) generated reactive oxygen species (ROS) in isolated mitochondria. Edaravone (10 - 100 microM) inhibited Ca(2+)-induced generation of ROS. These results suggest that edaravone inhibits opening of mitochondrial PTP in the brain, and they imply that inhibition of mitochondrial PTP may account for the neuroprotective effect of edaravone.

Nitric oxide-induced apoptosis in cultured rat astrocytes: protection by edaravone, a radical scavenger.

Nitric oxide induces apoptosis-like cell death in cultured astrocytes, but the exact mechanism is not known. This study further characterized the mechanism of nitric oxide-induced cytotoxicity, and examined the effect of edaravone, a radical scavenger, on cytotoxicity. Treatment of cultured rat astrocytes with sodium nitroprusside (SNP), a nitric oxide donor, for 72 h, decreased cell viability by causing apoptosis-like cell death. The injury was accompanied by increases in the production of reactive oxygen species and in the level of nuclear apoptosis-inducing factor, but not in caspase activity. SNP-induced cytotoxicity was blocked by the c-jun N-terminal protein kinase (JNK) inhibitor SP600125 (20 microM), the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (20 microM), and the extracellular signal-regulating kinase (ERK) inhibitor U0126 (10 microM), and the nitric oxide donor stimulated the phosphorylation of p38 MAP kinase, JNK, and ERK. Edaravone (10 microM) protected astrocytes against SNP-induced cell injury and it inhibited SNP-induced phosphorylation of p38 MAP kinase, JNK, and ERK, and the production of reactive oxygen species. Edaravone also attenuated SNP-induced increase in nuclear apoptosis-inducing factor levels. These results suggest that MAP kinase pathways play a key role in nitric oxide-induced apoptosis and that edaravone protects against nitric oxide-induced cytotoxicity by inhibiting nitric oxide-induced MAP kinase activation in astrocytes.

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody.

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.