Current Neuropharmacology

Published by Bentham Science Publishers
Online ISSN: 1570-159X
Sites on TRPV1 modulating function.  
Inflammatory mediators and intracellular signalling pathways modulating TRPV1.  
Injury or inflammation release a range of inflammatory mediators that increase the sensitivity of sensory neurons to noxious thermal or mechanical stimuli. The heat- and capsaicin-gated channel TRPV1, which is an important detector of multiple noxious stimuli, plays a critical role in the development of thermal hyperalgesia induced by a wide range of inflammatory mediators. We review here recent findings on the molecular mechanisms of sensitisation of TRPV1 by inflammatory mediators, including bradykinin, ATP, NGF and prostaglandins. We describe the signalling pathways believed to be involved in the potentiation of TRPV1, and our current understanding of how inflammatory mediators couple to these pathways.
Sex Related Differences in Stress-Induced Behavioral Deficits
Evidence that Facilitation of Serotonin Neurotransmission in the Hippocampus is Involved in Adaptation to Stress
Activity of male and female rats in an open field 24 h after a single (2h) or repeated (2h/day for 5 days) restraints. Male (body weights 200-250 gm) and female (body weights 190-230) animals were restrained for 2h on wire grids and activity in an open field was monitored 24 h after the termination of the 1st or 5th restraint period as described by Haleem & Parveen (1994). Values are means ± S. D. (n=6). Significant differences by Newman Keuls test: *P<0.01 from respective unrestrained animals; +P<0.01 from 1st day (2h/day) restrained animals, following two way ANOVA. (Haleem unpublished data).
Attenuation (red) and enhancement (green) of raphe-hippocampal serotonin neurotransmission regulated by somatodendritic 5-HT-1A receptors.
Stress is the major predisposing and precipitating factor in the onset of depression which is the most significant mental health risk for women. Behavioral studies in animal models show that female sex though less affected by an acute stressor; exposure to repeated stressors induces coping deficits to impair adaptation in them. A decrease in the function of 5-hydroxytryptamine (5-HT; serotonin) in the hippocampus and an increased function of the 5-HT-1A receptor in the raphe nucleus coexist in depression. Pharmacological and neurochemical data are relevant that facilitation of serotonin neurotransmission via hippocampus due to desensitization of somatodendritic 5-HT1A receptors may lead to adaptation to stress. The present article reviews research on sex related differences of raphe-hippocampal serotonin neurotransmission to find a possible answer that may account for the sex differences of adaptation to stress reported in preclinical research and greater incidence of depression in women than men.
Curcumin (diferuloylmethane), a polyphenol extracted from the plant Curcuma longa, is widely used in Southeast Asia, China and India in food preparation and for medicinal purposes. Since the second half of the last century, this traditional medicine has attracted the attention of scientists from multiple disciplines to elucidate its pharmacological properties. Of significant interest is curcumin's role to treat neurodegenerative diseases including Alzheimer's disease (AD), and Parkinson's disease (PD) and malignancy. These diseases all share an inflammatory basis, involving increased cellular reactive oxygen species (ROS) accumulation and oxidative damage to lipids, nucleic acids and proteins. The therapeutic benefits of curcumin for these neurodegenerative diseases appear multifactorial via regulation of transcription factors, cytokines and enzymes associated with (Nuclear factor kappa beta) NFκB activity. This review describes the historical use of curcumin in medicine, its chemistry, stability and biological activities, including curcumin's anti-cancer, anti-microbial, anti-oxidant, and anti-inflammatory properties. The review further discusses the pharmacology of curcumin and provides new perspectives on its therapeutic potential and limitations. Especially, the review focuses in detail on the effectiveness of curcumin and its mechanism of actions in treating neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and brain malignancies.
Chemical structures of MDMA, classical MDMA analogues and structurally related stimulants and hallucinogens.  
Chemical structures of a selection of classical phenylalkylamines described as possessing potential MDMA-like properties, as described in [121].  
Chemical structures of different MDMA and non-MDMA analogues of potential interest: brominated MDMA and MDA analogues (A), bioisosteres (B, C, D) and mescalinoids (E, F).  
Besides stimulants and hallucinogens, whose psychotropic effects are shared by many structurally related molecules exhibiting different efficacies and potencies in humans, the phenylisopropylamine MDMA (3,4-methylenedioxymethamphetamine, XTC, "Ecstasy") is the prototypical representative of a separate class of psychotropic substance, able to elicit the so-called entactogenic syndrome in healthy humans. This reversible altered state of consciousness, usually described as an "open mind state", may have relevant therapeutic applications, both in psychotherapy and as a pharmacological support in many neuropsychiatric disorders with a high rate of treatment failure. Nevertheless, a comprehensive and systematic exploration of the structure-activity relationships associated with entactogenic activity has remained incomplete and controversial, highlighting the possibility that MDMA might represent a pharmacological rarity in the field of psychotropics. As the latter is still an open question, the pharmacological characterization of MDMA analogues remains the logical strategy to attempt the elucidation of the structural requirements needed to elicit typical MDMA-like effects. Intriguingly, almost no experimental evidence supports the existence of actual MDMA analogues that truly resemble the whole pharmacological profile of MDMA, probably due to its complex (and partially not fully understood) mechanism of action that includes a disruption of monoaminergic neurotransmission. The present review presents a brief summary of the pharmacology of MDMA, followed by the evidence accumulated over the years regarding the characterization of classical structurally related MDMA analogues in different models and how this state of the art highlights the need to develop new and better MDMA analogues.
Genes that were Induced Both in the NAcc and FC in LEW Rats as Compared with F344 Rats
Genes that were Induced in the FC but not in the NAcc of LEW Rats as Compared with F344 Rats
Genes that were Inhibited both in the NAcc and FC of LEW Rats as Compared with F344 Rats
Genes that were Inhibited in the NAcc of LEW rats as Compared with F344 Rats
Drug addiction results from the interplay between social and biological factors. Among these, genetic variables play a major role. The use of genetically related inbred rat strains that differ in their preference for drugs of abuse is one approach of great importance to explore genetic determinants. Lewis and Fischer 344 rats have been extensively studied and it has been shown that the Lewis strain is especially vulnerable to the addictive properties of several drugs when compared with the Fischer 344 strain. Here, we have used microarrays to analyze gene expression profiles in the frontal cortex and nucleus accumbens of Lewis and Fischer 344 rats. Our results show that only a very limited group of genes were differentially expressed in Lewis rats when compared with the Fischer 344 strain. The genes that were induced in the Lewis strain were related to oxygen transport, neurotransmitter processing and fatty acid metabolism. On the contrary genes that were repressed in Lewis rats were involved in physiological functions such as drug and proton transport, oligodendrocyte survival and lipid catabolism. These data might be useful for the identification of genes which could be potential markers of the vulnerability to the addictive properties of drugs of abuse.
GAP-43 is an intracellular growth-associated protein that appears to assist neuronal pathfinding and branching during development and regeneration, and may contribute to presynaptic membrane changes in the adult, leading to the phenomena of neurotransmitter release, endocytosis and synaptic vesicle recycling, long-term potentiation, spatial memory formation, and learning. GAP-43 becomes bound via palmitoylation and the presence of three basic residues to membranes of the early secretory pathway. It is then sorted onto vesicles at the late secretory pathway for fast axonal transport to the growth cone or presynaptic plasma membrane. The palmitate chains do not serve as permanent membrane anchors for GAP-43, because at steady-state most of the GAP-43 in a cell is membrane-bound but is not palmitoylated. Filopodial extension and branching take place when GAP-43 is phosphorylated at Ser-41 by protein kinase C, and this occurs following neurotrophin binding and the activation of numerous small GTPases. GAP-43 has been proposed to cluster the acidic phospholipid phosphatidylinositol 4,5-bisphosphate in plasma membrane rafts. Following GAP-43 phosphorylation, this phospholipid is released to promote local actin filament-membrane attachment. The phosphorylation also releases GAP-43 from calmodulin. The released GAP-43 may then act as a lateral stabilizer of actin filaments. N-terminal fragments of GAP-43, containing 10-20 amino acids, will activate heterotrimeric G proteins, direct GAP-43 to the membrane and lipid rafts, and cause the formation of filopodia, possibly by causing a change in membrane tension. This review will focus on new information regarding GAP-43, including its binding to membranes and its incorporation into lipid rafts, its mechanism of action, and how it affects and is affected by extracellular agents.
Haplotype Analysis of HTR1B in Methamphetamine Dependence
Association of HTR1B with Clinical Phenotypes of Methamphetamine Dependence and Psychosis
Several lines of evidence implicate serotonergic dysfunction in diverse psychiatric disorders including anxiety, depression, and drug abuse. Mice with a knock-out of the 5HT1b receptor gene (HTR1B) displayed increased locomotor response to cocaine and elevated motivation to self-administer cocaine and alcohol. Previous genetic studies showed significant associations of HTR1B with alcohol dependence and substance abuse, but were followed by inconsistent results. We examined a case-control genetic association study of HTR1B with methamphetamine-dependence patients in a Japanese population. The subjects were 231 patients with methamphetamine dependence, 214 of whom had a co-morbidity of methamphetamine psychosis, and 248 age- and sex-matched healthy controls. The three single nucleotide polymorphisms (SNPs), rs130058 (A-165T), rs1228814 (A-700C) and rs1228814 (A+1180G) of HTR1B were genotyped. There was no significant difference in allelic and genotypic distributions of the SNPs between methamphetamine dependence and the control. Genetic associations of HTR1B were tested with several clinical phenotypes of methamphetamine dependence and/or psychosis, such as age at first abuse, duration of latency from the first abuse to onset of psychosis, prognosis of psychosis after therapy, and complication of spontaneous relapse of psychotic state. There was, however, no asscocation between any SNP and the clinical phenotypes. Haplotype analyses showed the three SNPs examined were within linkage disequilibrium, which implied that the three SNPs covered the whole HTR1B, and distribution of estimated haplotype frequency was not different between the groups. The present findings may indicate that HTR1B does not play a major role in individual susceptibility to methamphetamine dependence or development of methamphetamine-induced psychosis.
The possibility that pain perception and processing in the CNS results in cellular stress and may influence heat shock protein (HSP) expression was examined in a rat model of morphine dependence and withdrawal. Since activation of pain pathways result in exhaustion of growth factors, we examined the influence of cerebrolysin, a mixture of potent growth factors (BDNF, GDNF, NGF, CNTF etc,) on morphine induced HSP expression. Rats were administered morphine (10 mg/kg, s.c. /day) for 12 days and the spontaneous withdrawal symptoms were developed by cessation of the drug administration on day 13(th) that were prominent on day 14(th) and continued up to day 15(th) (24 to 72 h periods). In a separate group of rats, cerebrolysin was infused intravenously (5 ml/kg) once daily from day one until day 15(th). In these animals, morphine dependence and withdrawal along with HSP immunoreactivity was examined using standard protocol. In untreated group mild HSP immunoreaction was observed during morphine tolerance, whereas massive upregulation of HSP was seen in CNS during withdrawal phase that correlated well with the withdrawal symptoms and neuronal damage. Pretreatment with cerebrolysin did not affect morphine tolerance but reduced the HSP expression during this phase. Furthermore, cerebrolysin reduced the withdrawal symptoms on day 14(th) to 15(th). Taken together these observations suggest that cellular stress plays an important role in morphine induced pain pathology and exogenous supplement of growth factors, i.e. cerebrolysin attenuates HSP expression in the CNS and induce neuroprotection. This indicates a new therapeutic role of cerebrolysin in the pathophysiology of drugs of abuse, not reported earlier.
Primers Used in this Study
Genotypic and Allelic Distribution of the ADORA1 Gene SNPs in the METH Subjects and the Controls
Genotypic Distribution of the ADORA1 Gene SNPs in Subcategorized METH Subjects
Linkage Disequilibrium Mapping of the ADORA1 Gene
Several lines of evidence suggest that the dopaminergic nervous system contributes to methamphetamine (METH) dependence, and there is increasing evidence of antagonistic interactions between dopamine and adenosine receptors in METH abusers. We therefore hypothesized that variations in the A1 adenosine receptor (ADORA1) gene modify genetic susceptibility to METH dependence/psychosis. In this study, we identified 7 single nucleotide polymorphisms (SNPs) in exons and exon-intron boundaries of the ADORA1 gene in a Japanese population. A total of 171 patients and 229 controls were used for an association analysis between these SNPs and METH dependence/psychosis. No significant differences were observed in either the genotypic or allelic frequencies between METH dependent/psychotic patients and controls. A global test of differentiation among samples based on haplotype frequencies showed no significant association. In the clinical feature analyses, no significant associations were observed among latency of psychosis, prognosis of psychosis, and spontaneous relapse. These results suggest that the ADORA1 gene variants may make little or no contribution to vulnerability to METH dependence/psychosis.
The adenosine A(2A) receptor (A(2A)R) is in the center of a neuromodulatory network affecting a wide range of neuropsychiatric functions by interacting with and integrating several neurotransmitter systems, especially dopaminergic and glutamatergic neurotransmission. These interactions and integrations occur at multiple levels, including (1) direct receptor- receptor cross-talk at the cell membrane, (2) intracellular second messenger systems, (3) trans-synaptic actions via striatal collaterals or interneurons in the striatum, (4) and interactions at the network level of the basal ganglia. Consequently, A(2A)Rs constitute a novel target to modulate various psychiatric conditions. In the present review we will first summarize the molecular interaction of adenosine receptors with other neurotransmitter systems and then discuss the potential applications of A(2A)R agonists and antagonists in physiological and pathophysiological conditions, such as psychostimulant action, drug addiction, anxiety, depression, schizophrenia and learning and memory.
Potential periphery to brain links between type 1 and type 2 diabetes and Alzheimer's disease. Peripheral insulin deficiency or insulin resistance leading to hyperglycemia, increased deposition of advanced glycation endproduct (AGE) modified proteins in the vasculature and increased vascular inflammation. These events lead to reduced transport of insulin across the blood brain barrier to the brain. Reduced neuronal insulin receptor signaling, due to insulin deficiency or insulin receptor desensitization alter a number of cellular signaling pathways that can promote amyloid beta (A) formation, phosphorylation of tau and reduced glucose uptake and metabolism by neurons.  
Consequences of insulin and A interactions on reduced neuronal insulin receptor signaling and promoting Alzheimer's disease pathology. In type 2 diabetes, there can be decreased or increased levels of insulin in brain (depending on disease state), along with insulin receptor desensitization. A peptide levels can be enhanced by reduced insulin receptor signaling, and soluble A oligomers can also block these receptors. Increased A levels will compete for insulin degrading enzyme with cerebral insulin. A aggregates can also have direct membrane toxic effect on neuronal cells. Reduced insulin receptor signaling results in reduced PI-3K activity that leads to reduced PKB/AKT activity. The consequences of this include reduced glucose metabolism and increased oxidative stress. Specifically reduced GSK3 phosphorylation leads to increased tau phosphorylation and A formation. Abbreviations: PI-3K-phosphoinositide-3 kinase: PKB/AKT – protein kinase B; GSK3 – glycogen synthase kinase 3: A amyloid beta peptide.  
There is an urgent need for new ways to treat Alzheimer's disease (AD), the most common cause of dementia in the elderly. Current therapies are modestly effective at treating the symptoms, and do not significantly alter the course of the disease. Over the years, a range of epidemiological and experimental studies have demonstrated interactions between diabetes mellitus and AD. As both diseases are leading causes of morbidity and mortality in the elderly and are frequent co-morbid conditions, it has raised the possibility that treating diabetes might be effective in slowing AD. This is currently being attempted with drugs such as the insulin sensitizer rosiglitazone. These two diseases share many clinical and biochemical features, such as elevated oxidative stress, vascular dysfunction, amyloidogenesis and impaired glucose metabolism suggesting common pathogenic mechanisms. The main thrust of this review will be to explore the evidence from a pathological point of view to determine whether diabetes can cause or exacerbate AD. This was supported by a number of animal models of AD that have been shown to have enhanced pathology when diabetic conditions were induced. The one drawback in linking diabetes and insulin to AD has been the postmortem studies of diabetic brains demonstrating that AD pathology was not increased; in fact decreased pathology has often been reported. In addition, diabetes induces its own distinct features of neuropathology different from AD. There are common pathological features to be considered including vascular abnormalities, a major feature arising from diabetes; there is increasing evidence that vascular abnormalities can contribute to AD. The most important common mechanism between insulin-resistant (type II) diabetes and AD could be impaired insulin signaling; a form of toxic amyloid can damage neuronal insulin receptors and affect insulin signaling and cell survival. It has even been suggested that AD could be considered as "type 3 diabetes" since insulin can be produced in brain. Another common feature of diabetes and AD are increased advanced glycation endproduct-modified proteins are found in diabetes and in the AD brain; the receptor for advanced glycation endproducts plays a prominent role in both diseases. In addition, a major role for insulin degrading enzyme in the degradation of Aβ peptide has been identified. Although clinical trials of certain types of diabetic medications for treatment of AD have been conducted, further understanding the common pathological processes of diabetes and AD are needed to determine whether these diseases share common therapeutic targets.
Connectivity of the NAc with other brain regions such as prefrontal cortex, ventral tegmental area (VTA). Activity of the NAc is mainly regulated by dopaminergic (from VTA) and glutamatergic afferents (from frontal cortex, amygdala and hippocampus ). In addition, inputs of GABAergic, serotonergic (from raphe) and adrenergic afferents (from locus ceruleus) as well as cholinergic interneuons (within the NAc) also modified activity of the NAc.  
Schematic illustration of connections of the NAc shell and core regions with subregions of hippocampus and prefrontal cortex. Please note that three portions of the NAc could be affected efficiently by two regions of hippocampus and prefrontal cortex.  
Schema of regulation of GABAergic medium spiny projecting neurons and the relationships among dopamine, glutamate, GABA, acetylcholine, enkephaline, dynorphin and their receptors in the NAc. Activity of GABAergic medium spiny neurons in the NAc is heavily regulated by dopaminergic (from ventral tegmental area) and glutamatergic afferents (from frontal cortex, amygdala and hippocampus) as well as GABAergic and cholinergic interneuons. Dopaminergic and glutamatergic activity in the NAc is also regulated by neuropeptides such as dynorphin and enkephalin. Regulation by other neurotransmitters such as serotonin and neuropeptides such as MCH and CART peptides may be involved.  
There is accumulating evidence that the nucleus accumbens (NAc) plays an important role in the pathophysiology of depression. Given that clinical depression is marked by anhedonia (diminished interest or pleasure), dysfunction of the brain reward pathway has been suggested as contributing to the pathophysiology of depression.Since the NAc is the center of reward and learning, it is hypothesized that anhedonia might be produced by hampering the function of the NAc. Indeed, it has been reported that stress, drug exposure and drug withdrawal, all of which produce a depressive-phenotype, alter various functions within the NAc, leading to inhibited dopaminergic activity in the NAc.In this review, we describe various factors as possible candidates within the NAc for the initiation of depressive symptoms. First, we discuss the roles of several neurotransmitters and neuropeptides in the functioning of the NAc, including dopamine, glutamate, gamma-aminobutyric acid (GABA), acetylcholine, serotonin, dynorphin, enkephaline, brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), melanin-concentrating hormone (MCH) and cocaine- and amphetamine-regulated transcript (CART). Second, based on previous studies, we propose hypothetical relationships among these substances and the shell and core subregions of the NAc.
Examples of brain MR imaging in NBIA disorders; showing a case of pantothenate kinase associated neurodegeneration (PKAN) (left), Kufor Rakeb disease (due to ATP13A2 mutations) (center) and neuroferritinopathy (due to FTL mutations) (right). In PKAN there is a classic eye of the tiger sign. Iron accumulation affects the putamen and caudate in our Kufor Rakeb disease patient. In this gene-proven neuroferritinopathy patient there is iron deposition in the basal ganglia, with a slight hint of thalamic involvement. Reproduced from [3]. 
Overview of NBIA Conditions and Genes (if Known)
Comparison of Aceruloplasminemia and Neuroferritinopathy
Reported Cases of Brain Lesioning and Deep Brain Stimulation in NBIA Disorders
Our understanding of the syndromes of Neurodegeneration with Brain Iron Accumulation (NBIA) continues to grow considerably. In addition to the core syndromes of pantothenate kinase-associated neurodegeneration (PKAN, NBIA1) and PLA2G6-associated neurodegeneration (PLAN, NBIA2), several other genetic causes have been identified (including FA2H, C19orf12, ATP13A2, CP and FTL). In parallel, the clinical and pathological spectrum has broadened and new age-dependent presentations are being described. There is also growing recognition of overlap between the different NBIA disorders and other diseases including spastic paraplegias, leukodystrophies and neuronal ceroid lipofuscinosis which makes a diagnosis solely based on clinical findings challenging. Autopsy examination of genetically-confirmed cases demonstrates Lewy bodies, neurofibrillary tangles, and other hallmarks of apparently distinct neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease. Until we disentangle the various NBIA genes and their related pathways and move towards pathogenesis-targeted therapies, the treatment remains symptomatic. Our aim here is to provide an overview of historical developments of research into iron metabolism and its relevance in neurodegenerative disorders. We then focus on clinical features and investigational findings in NBIA and summarize therapeutic results reviewing reports of iron chelation therapy and deep brain stimulation. We also discuss genetic and molecular underpinnings of the NBIA syndromes.
Depressor effect of brain ACE2-Ang-(1-7)-Mas axis and the related mechanisms. Abbreviations: ACE, Angiotensin converting
enzyme; Ang I, Angiotensin I; Ang II, Angiotensin II; Ang-(1-7), Angiotensin-(1-7); AT1R , Angiotensin II type 1 receptor; AT2R ,
Angiotensin II type 2 receptor; BK, Bradykinin; eNOS, Endothelial nitric oxide synthase; IKv, Delayed rectifier K+ current; IL-1β,
Interleukin-1β; IL-6, Interleukin-6; MDA, Malondialdehyde; NE, Norepinephrine; nNOS, Neuronal nitric oxide synthase; NO, Nitric oxide;
SOD, Super oxygen dehydrogenises; TNF-α, Tumour necrosis factor–α.
Anti-atherosclerotic and antithrombotic actions of ACE2-Ang-(1-7)-Mas axis. Abbreviations: Ang-(1-7), Angiotensin-(1-7); AT2R ,
Angiotensin II type 2 receptor; BK, Bradykinin; eNOS, Endothelial nitric oxide synthase; ERK, Extracellular signal-regulated kinase; HO-1,
Heme oxygenase-1; LOX-1, Lectin-like oxidized low-density lipoprotein receptor-1; MCP-1, Monocyte chemoattractant protein-1; NO,
Nitric oxide; P38 MAPK, P38 mitogen-activated protein kinase; PCNA, Proliferating cell nuclear antigen; PGI2, Prostaglandin I2; ROS,
Reactive oxygen species; VSMC, Vascular smooth muscle cell.
Neuroprotective effect of brain ACE2-Ang-(1-7)-Mas axis after ischemia insult. Abbreviations: Ang-(1-7), Angiotensin-(1-7); BK,
Bradykinin; COX-2, Cyclooxygenase-2; eNOS, Endothelial nitric oxide synthase; IL-1β, Interleukin-1β; iNOS, Inducible nitric oxide
synthase; MDA, Malondialdehyde; NF-κB, Nuclear actor-κB. NO, Nitric oxide; SOD, Super oxygen dehydrogenises; TNF-α, Tumour ecrosis
The renin-angiotensin system (RAS) in brain is a crucial regulator for physiological homeostasis and diseases of cerebrovascular system, such as ischemic stroke. Overactivation of brain Angiotensin-converting enzyme (ACE) - Angiotensin II (Ang II) - Angiotensin II type 1 receptor (AT1R) axis was found to be involved in the progress of hypertension, atherosclerosis and thrombogenesis, which increased the susceptibility to ischemic stroke. Besides, brain Ang II levels have been revealed to be increased in ischemic tissues after stroke, and contribute to neural damage through elevating oxidative stress levels and inducing inflammatory response in the ischemic hemisphere via AT1R. In recent years, new components of RAS have been discovered, including ACE2, Angiotensin-(1-7) [Ang-(1-7)] and Mas, which constitute ACE2-Ang-(1-7)-Mas axis. ACE2 converts Ang II to Ang-(1-7), and Ang-(1-7) binds with its receptor Mas, exerting benefical effects in cerebrovascular disease. Through interacting with nitric oxide and bradykinin, Ang-(1-7) could attenuate the development of hypertension and the pathologic progress of atherosclerosis. Besides, its antithrombotic activity also prevents thrombogenic events, which may contribute to reduce the risk of ischemic stroke. In addition, after ischemia insult, ACE2-Ang-(1-7)-Mas has been shown to reduce the cerebral infarct size and improve neurological deficits through its antioxidative and anti-inflammatory effects. Taken together, activation of the ACE2-Ang-(1-7)-Mas axis may become a novel therapeutic target in prevention and treatment of ischemia stroke, which deserves further investigations.
Effect of 3-nitropropionic acid (3-NPA) and acetyl-Lcarnitine (ALC) on Il10 gene expression in PC12 cells. Mean ± SEM.
Effect of 3-nitropropionic acid (3-NPA) and acetyl-Lcarnitine (ALC) on Ptgs1 (Cox1) gene expression in PC12 cells. Mean ± SEM.
The neurotoxicity induced by the mitochondrial inhibitor 3-nitropropionic acid (3-NPA) is associated with a decrease of ATP synthesis and an increase of free radical production which can lead to apoptosis or necrosis. We have used the PC12, neuron-like rat pheochromocytoma cell line, to study further the mechanism of 3-NPA-evoked neurotoxicity and the effects of acetyl-L-carnitine (ALC) which has neuroprotective actions against various types of mitochondrial inhibitors. Cultured PC 12 cells were exposed to a low dose of 3-NPA 50 (microM) in the presence or absence of 5 mM ALC. The dose of 3-NPA was sub toxic and no changes in pro-apoptotic Bax or anti-apoptotic Bcl-2 gene expression were observed. We followed specific genetic markers to look for changes evoked by 3-NPA toxicity and also changes associated with neuroprotection exerted by the ALC treatment, using RT-PCR arrays (delta-delta method). 3-NPA exposure evoked a decrease in expression of the Tp53 gene. This down regulation was prevented by pretreatment of the cells with ALC. The Tp53 gene responds to cellular stresses and the effects seen here are possibly associated with the 3-NPA evoked changes in mitochondrial metabolism. Other genes associated with stress and apoptosis, Parp-1, Bcl-2, and Bax were not affected by 3-NPA or ALC. The decrease of inflammatory response Il-10 gene expression due to 3-NPA was further lowered by presence of ALC. Other inflammation related genes, Il1rn, Nr3c1 and Cxcr4 were not affected. Interestingly, the glutamate transporter slc17a7, carnitine-acylcarnitine translocase Slc25a20 and heat shock proteins genes, Hsp27, Hmox1 (Hsp32, HO1) as well as Hspa 1a (Hsp 70) increased only when both ALC and small dose of 3-NPA were present. The alterations in gene expression detected in this study suggest role of several intracellular pathways in the neurotoxicity of 3-NPA and the neuroprotection against 3-NPA-induced neurotoxicity by ALC.
During the past two decades, many pharmacological strategies have been investigated for the management of painful neuropathies. However, neuropathic pain still remains a clinical challenge. A combination of therapies is often required, but unfortunately in most cases adequate pain relief is not achieved. Recently, attention has been focused on the physiological and pharmacological effects of L-acetylcarnitine in neurological disorders. There are a number of reports indicating that L-acetylcarnitine can be considered as a therapeutic agent in neuropathic disorders including painful peripheral neuropathies. In this review article, we will examine the antinociceptive and the neuroprotective effects of Lacetylcarnitine as tested in clinical studies and in animal models of nerve injury.
Muscarinic acetylcholine receptors (mAChRs) are prototypical Family A G protein coupled-receptors. The five mAChR subtypes are widespread throughout the periphery and the central nervous system and, accordingly, are widely involved in a variety of both physiological and pathophysiological processes. There currently remains an unmet need for better therapeutic agents that can selectively target a given mAChR subtype to the relative exclusion of others. The main reason for the lack of such selective mAChR ligands is the high sequence homology within the acetylcholine-binding site (orthosteric site) across all mAChRs. However, the mAChRs possess at least one, and likely two, extracellular allosteric binding sites that can recognize small molecule allosteric modulators to regulate the binding and function of orthosteric ligands. Extensive studies of prototypical mAChR modulators, such as gallamine and alcuronium, have provided strong pharmacological evidence, and associated structure-activity relationships (SAR), for a "common" allosteric site on all five mAChRs. These studies are also supported by mutagenesis experiments implicating the second extracellular loop and the interface between the third extracellular loop and the top of transmembrane domain 7 as contributing to the common allosteric site. Other studies are also delineating the pharmacology of a second allosteric site, recognized by compounds such as staurosporine. In addition, allosteric agonists, such as McN-A-343, AC-42 and N-desmethylclozapine, have also been identified. Current challenges to the field include the ability to effectively detect and validate allosteric mechanisms, and to quantify allosteric effects on binding affinity and signaling efficacy to inform allosteric modulator SAR.
Acetylcholinesterase (AChE) (EC is an important enzyme that breaks down of acetylcholine in synaptic cleft in neuronal junctions. Inhibition of AChE is associated with treatment of several diseases such as Alzheimer's disease (AD), myasthenia gravis, and glaucoma as well as the mechanisms of insecticide and anthelmintic drugs. Several AChE inhibitors are available in clinical use currently for the treatment of AD; however, none of them has ability, yet, to seize progress of the disease. Consequently, an extensive research has been going on finding new AChE inhibitors. In this sense, natural inhibitors have gained great attention due to their encouraging effects toward AChE. In this review, promising candidate molecules with marked AChE inhibition from both plant and animal sources will be underlined.
As acetylcholinesterase (AChE) inhibitors are an important therapeutic strategy in Alzheimer’s disease, efforts are being made in search of new molecules with anti-AChE activity. The fact that naturally-occurring compounds from plants are considered to be a potential source of new inhibitors has led to the discovery of an important number of secondary metabolites and plant extracts with the ability of inhibiting the enzyme AChE, which, according to the cholinergic hypothesis, increases the levels of the neurotransmitter acetylcholine in the brain, thus improving cholinergic functions in patients with Alzheimer’s disease and alleviating the symptoms of this neurological disorder. This review summarizes a total of 128 studies which correspond to the most relevant research work published during 2006-2012 (1st semester) on plant-derived compounds, plant extracts and essential oils found to elicit AChE inhibition.
Flowchart of the selection of studies. After reading the title and the abstract we excluded the articles that did not meet the inclusion criteria. After reading the whole article we excluded two of them which did not used supplements, but only changed the diet of the participants; one which did not have a group with ADHD alone, and three which were not randomized.  
Since attention deficit/hyperactivity disorder (ADHD) presents high prevalence among children, science has been researching alternative forms of treatment that do not involve medication. To evaluate the effects of polyunsaturated fatty acids (PUFAs) on attention deficit/hyperactivity disorder. We reviewed the articles published between 1980 and 2012 indexed in the databases PubMed, APA psychNET, Scopus and Web of Knowledge. Initially 231 articles were selected, out of which 12 met the inclusion criteria. The articles selected reported a modest cognitive and behavioral improvement of the patients after treatment with low doses of PUFAs. Those results might be associated with the evaluation methodology, the doses of PUFAs administered or the duration of treatment.
Affinities of some Atypical Antipsychotic Agents at D1 and D2 Dopamine Receptors, and at M1 and M4 Muscarinic Receptors 
Alternative hypotheses for indirect action of dopamine D2-receptor-blocking antipsychotic drugs. Both hypotheses postulate increased firing of striatal cholinergic interneurons, and that the final common target of antipsychotic drugs is reduced formation of cAMP. Left: Indirect action mediated by the aversive effects of extrapyramidal side effects (EPS). Right: Indirect action mediated by increased activation of striatal muscarinic M4 receptors. 
Many issues remain unresolved about antipsychotic drugs. Their therapeutic potency scales with affinity for dopamine D2 receptors, but there are indications that they act indirectly, with dopamine D1 receptors (and others) as possible ultimate targets. Classical neuroleptic drugs disinhibit striatal cholinergic interneurones and increase acetyl choline release. Their effects may then depend on stimulation of muscarinic receptors on principle striatal neurones (M4 receptors, with reduction of cAMP formation, for therapeutic effects; M1 receptors for motor side effects). Many psychotic patients do not benefit from neuroleptic drugs, or develop resistance to them during prolonged treatment, but respond well to clozapine. For patients who do respond, there is a wide (>ten-fold) range in optimal doses. Refractoriness or low sensitivity to antipsychotic effects (and other pathologies) could then arise from low density of cholinergic interneurones. Clozapine probably owes its special actions to direct stimulation of M4 receptors, a mechanism available when indirect action is lost.
Potential Clozapine-like Drugs: Synopsis 
Studies Giving Individual Data on Plasma Levels and Clinical Response, for Haloperidol in Treatment of Acute Psychosis 
Rapid-onset psychotic rebound is uncommon on discontinuation of most antipsychotic drugs, as might be expected for antipsychotic drugs with (hypothetically) indirect actions at their final target receptors. Rapid-onset psychosis is more common on withdrawal of clozapine, which might be expected if its action is direct. Drugs other than clozapine (notably thioridazine) may have hitherto unrecognised similarities to clozapine (but without danger of agranulocytosis), and may be useful in treatment of refractory psychosis. Quetiapine fulfils only some criteria for a clozapine-like drug. Clinical response to neuroleptics varies widely at any given plasma level. Haase's "neuroleptic threshold" concept suggests that the dose producing the slightest motor side effects produces most or all of the therapeutic benefit, but analyses presented here suggest that antipsychotic actions are not subject to a sharp "all-or-none" threshold but increase over a small dose range. This concept could provide a method for quantitative determination of individualized optimal doses.
Intravenous immunoglobulins (IVIg) have been shown in a number of trials, to be an effective treatment for the three main types of inflammatory neuropathies: Guillain-Barré Syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and multifocal motor neuropathy (MMN). IVIg is thought to exert its immunomodulatory effects by affecting several components of the immune system including B-cells, T-cells, macrophages, complement, cytokines and cellular adhesion molecules. This article reviews the published evidence and the principal postulated mechanisms of action of intravenous immunoglobulins with special emphasis on inflammatory neuropathies.
A schematic view of SN and VTA connections. DA neurons receive input from the local GABAergic interneurons and input from other brain areas. Both areas receive serotonergic input from the dorsal raphe nucleus (DRN). The dotted line indicates the subdivision of the SN: the SN pars reticulata (SN r ) and the SN pars compacta (SN c ). Neurons receiving a collective input are grouped (dotted circles). GABAergic, glutamatergic and serotonergic inputs are indicated by black, white and gray symbols, respectively. HIPP, hippocampus; NAC, nucleus accumbens; PFC, prefrontal cortex; STN, subthalamic nucleus.  
Model scheme for atypical antipsychotic drug action. The two schemes above represent the situation under normal conditions, where activation of 5-HT receptors (an unknown (5- HT X ) receptor on SN and the 5-HT 2 receptor on VTA DA neurons) can enhance the DA D 2 receptor-mediated autoinhibitory process. The two schemes below represent the situation in the presence of an atypical antipsychotic drug. This drug antagonizes a portion of the DA D 2 receptors, thereby reducing GIRK channel function, resulting in depolarization and increased firing activity of the DA neurons. Prolonged depolarization could, theoretically, lead to the induction of depolarization blockade and subsequently therapeutic efficacy and extra-pyramidal side-effects (EPS). In VTA DA neurons an atypical antipsychotic drug will, by blocking the 5-HT 2 receptors, prevent 5-HT 2 -mediated enhancement of GIRK channels, and thus auto-inhibition. This would permit a further depolarization, and ultimately depolarization blockade. In SN DA neurons " normal " enhancement of auto-inhibition can occur through the 5-HT X receptor (which is not affected by the antipsychotic drug), a process that will reduce the chance that depolarization blockade will develop.  
Schizophrenia has been associated with a dysfunction of brain dopamine (DA). This, so called, DA hypothesis has been refined as new insights into the pathophysiology of schizophrenia have emerged. Currently, dysfunction of prefrontocortical glutamatergic and GABAergic projections and dysfunction of serotonin (5-HT) systems are also thought to play a role in the pathophysiology of schizophrenia. Refinements of the DA hypothesis have lead to the emergence of new pharmacological targets for antipsychotic drug development. It was shown that effective antipsychotic drugs with a low liability for inducing extra-pyramidal side-effects have affinities for a range of neurotransmitter receptors in addition to DA receptors, suggesting that a combination of neurotransmitter receptor affinities may be favorable for treatment outcome.This review focuses on the interaction between DA and 5-HT, as most antipsychotics display affinity for 5-HT receptors. We will discuss DA/5-HT interactions at the level of receptors and G protein-coupled potassium channels and consequences for induction of depolarization blockade with specific attention to DA neurons in the ventral tegmental area (VTA) and the substantia nigra zona compacta (SN), neurons implicated in treatment efficacy and the side-effects of schizophrenia, respectively. Moreover, it has been reported that electrophysiological interactions between DA and 5-HT show subtle, but important, differences between the SN and the VTA which could explain (in part) the effectiveness and lower propensity to induce side-effects of the newer atypical antipsychotic drugs. In that respect the functional implications of DA/5-HT interactions for schizophrenia will be discussed.
Prostaglandins, in particular PGE(2) and prostacyclin PGI(2) have diverse biological effects. Most importantly, they are involved in inflammation and pain. Prostaglandins in nano- and micromolar concentrations sensitize nerve cells, i.e. make them more sensitive to electrical or chemical stimuli. Sensitization arises from the effect of prostaglandins on ion channels and occurs both at the peripheral terminal of nociceptors at the site of tissue injury (peripheral sensitization) and at the synapses in the spinal cord (central sensitization). The first step is the binding of prostaglandins to receptors in the cell membrane, mainly EP and IP receptors. The receptors couple via G proteins to enzymes such as adenylate cyclase and phospholipase C (PLC). Activation of adenylate cyclase leads to increase of cAMP and subsequent activation of protein kinase A (PKA) or PKA-independent effects of cAMP, e.g. mediated by Epac (=exchange protein activated by cAMP). Activation of PLC causes increase of inositol phosphates and increase of cytosolic calcium. This article summarizes the effects of PGE(2), PGE(1), PGI2 and its stable analogues on non-selective cation channels and sodium, potassium, calcium and chloride channels. It describes the mechanism responsible for the facilitatory or inhibitory prostaglandin effects on ion channels. Understanding these mechanisms is essential for the development of useful new analgesics.
gamma-aminobutyric acid (GABA) plays important roles in the central nervous system, acting as a neurotransmitter on both ionotropic ligand-gated Cl(-)-channels, and metabotropic G-protein coupled receptors (GPCRs). These two types of receptors called GABA(A) (and C) and GABA(B) are the targets of major therapeutic drugs such as the anxiolytic benzodiazepines, and antispastic drug baclofen (lioresal(R)), respectively. Although the multiplicity of GABA(A) receptors offer a number of possibilities to discover new and more selective drugs, the molecular characterization of the GABA(B) receptor revealed a unique, though complex, heterodimeric GPCR. High throughput screening strategies carried out in pharmaceutical industries, helped identifying new compounds positively modulating the activity of the GABA(B) receptor. These molecules, almost devoid of apparent activity when applied alone, greatly enhance both the potency and efficacy of GABA(B) agonists. As such, in contrast to baclofen that constantly activates the receptor everywhere in the brain, these positive allosteric modulators induce a large increase in GABA(B)-mediated responses only WHERE and WHEN physiologically needed. Such compounds are then well adapted to help GABA to activate its GABA(B) receptors, like benzodiazepines favor GABA(A) receptor activation. In this review, the way of action of these molecules will be presented in light of our actual knowledge of the activation mechanism of the GABA(B) receptor. We will then show that, as expected, these molecules have more pronounced in vivo responses and less side effects than pure agonists, offering new potential therapeutic applications for this new class of GABA(B) ligands.
Oxygen and the continuity of life through the most suitable energy production (right). On the left side: metabolism, ageing and pathological conditions involving free radicals and oxidative stress.
Melatonin effects on cytoskeletal alterations produced by okadaic acid (OA) or hydrogen peroxide (H2O2) in N1E-115 cells. (A) N1E-115 cells were incubated with the vehicle (VEH), 15 nM OA for 24 h (OA), 100 µM H2O2 for 1 h (H2O2), 0.1 µM melatonin for 6 h before treatment with 15 nM okadaic acid for 24 h (MEL+AO) or 0.1 µM melatonin for 3 h before 1 h treatment with 100 µM H2O2 (MEL+ H2O2) Cultures were fixed and simultaneously stained with an antitubulin antibody (green), RITC-phalloidin (red) and DAPI for detection of microtubules, actin microfilaments and the nucleus, respectively. Bar = 10 mm. (B) Melatonin effects on tau hyperphosphorylation induced by OA in N1E-115 cells. 1) Melatonin effects in phospho-tau levels were analyzed by Western blot. Cells were incubated with the VEH, OA and MEL+OA. Representative autoradiogram of p-tau in the VEH, OA, or MEL+OA. 2) Optical density (mm2) of p-tau immunoreactivity is showed in the histogram. Results represent the mean ± S.E.M. of three experiments done in quadruplicate. (C) Melatonin effect on lipid per-oxidation induced by OA and H2O2. Neuroblastoma cells were incubated with the VEH, H2O2, MEL+ H2O2, OA and MEL+OA. Cells were homogenized and both MDA and 4-HDA were quantified. Results represent the mean ± SEM of three experiments done in duplicate.
Molecular oxygen is toxic for anaerobic organisms but it is also obvious that oxygen is poisonous to aerobic organisms as well, since oxygen plays an essential role for inducing molecular damage. Molecular oxygen is a triplet radical in its ground-stage (.O-O.) and has two unpaired electrons that can undergoes consecutive reductions of one electron and generates other more reactive forms of oxygen known as free radicals and reactive oxygen species. These reactants (including superoxide radicals, hydroxyl radicals) possess variable degrees of toxicity. Nitric oxide (NO•) contains one unpaired electron and is, therefore, a radical. NO• is generated in biological tissues by specific nitric oxide synthases and acts as an important biological signal. Excessive nitric oxide production, under pathological conditions, leads to detrimental effects of this molecule on tissues, which can be attributed to its diffusion-limited reaction with superoxide to form the powerful and toxic oxidant, peroxynitrite. Reactive oxygen and nitrogen species are molecular “renegades”; these highly unstable products tend to react rapidly with adjacent molecules, donating, abstracting, or even sharing their outer orbital electron(s). This reaction not only changes the target molecule, but often passes the unpaired electron along to the target, generating a second free radical, which can then go on to react with a new target amplifying their effects. This review describes the mechanisms of oxidative damage and its relationship with the most highly studied neurodegenerative diseases and the roles of melatonin as free radical scavenger and neurocytoskeletal protector.
The endocannabinoid signaling system is composed of the cannabinoid receptors; their endogenous ligands, the endocannabinoids; the enzymes that produce and inactivate the endocannabinoids; and the endocannabinoid transporters. The endocannabinoids are a new family of lipidic signal mediators, which includes amides, esters, and ethers of long-chain polyunsaturated fatty acids. Endocannabinoids signal through the same cell surface receptors that are targeted by Delta(9)-tetrahydrocannabinol (Delta(9)THC), the active principles of cannabis sativa preparations like hashish and marijuana. The biosynthetic pathways for the synthesis and release of endocannabinoids are still rather uncertain. Unlike neurotransmitter molecules that are typically held in vesicles before synaptic release, endocannabinoids are synthesized on demand within the plasma membrane. Once released, they travel in a retrograde direction and transiently suppress presynaptic neurotransmitter release through activation of cannabinoid receptors. The endocannabinoid signaling system is being found to be involved in an increasing number of pathological conditions. In the brain, endocannabinoid signaling is mostly inhibitory and suggests a role for cannabinoids as therapeutic agents in central nervous system (CNS) disease. Their ability to modulate synaptic efficacy has a wide range of functional consequences and provides unique therapeutic possibilities. The present review is focused on new information regarding the endocannabinoid signaling system in the brain. First, the structure, anatomical distribution, and signal transduction mechanisms of cannabinoid receptors are described. Second, the synthetic pathways of endocannabinoids are discussed, along with the putative mechanisms of their release, uptake, and degradation. Finally, the role of the endocannabinoid signaling system in the CNS and its potential as a therapeutic target in various CNS disease conditions, including alcoholism, are discussed.
Examples of chloride currents for α 1 β 2 γ 2s , α 5 β 2 γ 2s , and α 6 β 2 γ 2s GABA A receptors. Left panels show representative whole-cell recordings achieved by applications of increasing concentrations of GABA (black bars). Right panels show paired recordings with the same concentrations of GABA in the presence of 2 MAC isoflurane (blue bar). GABA concentrations (all in µM) for α 1 β 2 γ 2s GABA A receptors (0.3, 1, 3, 10, 30, 100, 300, 1000), for α 5 β 2 γ 2s GABA A receptors (0.1, 0.3, 1, 3, 10, 30, 100, 300), and for α 6 β 2 γ 2s GABA A receptors (0.1, 0.3, 1, 3, 10, 30, 100). Vertical scale bars 100 pA; Horizontal scale bars 10 s. The data indicate that α 6 containing receptors are more sensitive to low concentrations of anesthetic than α 1 β 2 γ 2s , or α 5 β 2 γ 2s receptors and that isoflurane is more effective at reducing the apparent affinity of GABA at α 5 containing receptors than at α 1 β 2 γ 2s , or α 6 β 2 γ 2s GABA A receptors. The methods used to collect these results have been described previously [59].  
General anesthetic drugs interact with many receptors in the nervous system, but only a handful of these interactions are critical for producing anesthesia. Over the last 20 years, neuropharmacologists have revealed that one of the most important target sites for general anesthetics is the GABA(A) receptor. In this review we will discuss what is known about anesthetic - GABA(A) receptor interactions.
The causal role of ammonium in hepatic encephalopathy was identified in 1930s. Astroglial cells are primary cellular elements of hepatic encephalopathy which conceptually, can be considered a toxic astrogliopathology. Previously we have reported that acute exposure to ammonium activated ouabain/Na,K-ATPase signalling pathway, which includes Src, EGF receptor, Raf, Ras, MEK and ERK1/2. Chronic incubation of astrocytes with ammonium increased production of endogenous ouabain-like compound. Ouabain antagonist canrenone abolished effects of ammonium on astrocytic swelling, ROS production, and upregulation of gene expression and function of TRPC1 and Cav1.2. However, ammonium induces multiple pathological modifications in astrocytes, and some of them may be not related to this signalling pathway. In this review, we focus on the effect of ammonium on ouabain/Na,K-ATPase signalling pathway and its involvement in ammonium-induced ROS production, cell swelling and aberration of Ca(2+) signals in astrocytes. We also briefly discuss Na,K-ATPase, EGF receptor, endogenous ouabain and ouabain antagonist.
Current Responses Induced by the Selected PF Compound Pools in the First Screening Step. Normalized current responses to the pools of PF compounds by the response to BaCl 2. PF 9-PF 12, PF 401-PF 404, and PF 409-PF 412 pools were selected as candidate agonists, and PF 37-PF 40, PF 157-PF 160, PF 185-PF 188, PF 233-PF 236, and PF 245-PF 248 pools were selected as candidate antagonists. The PF 417-PF 420 pool was selected as a candidate agonist or antagonist in the first screening step.
Current Responses Induced by the Selected PF Compound Pools in the First Screening Step. Normalized current responses to the pools of PF compounds by the response to BaCl2. PF 9 – PF 12, PF 401 – PF 404, and PF 409 – PF 412 pools were selected as candidate agonists, and PF 37 – PF 40, PF 157 – PF 160, PF 185 – PF 188, PF 233 – PF 236, and PF 245 – PF 248 pools were selected as candidate antagonists. The PF 417 – PF 420 pool was selected as a candidate agonist or antagonist in the first screening step.
Candidate Agonist and Antagonists Identified in the Second Screening Step. PF 419 was selected as a candidate agonist, and PF 40, PF 236, and PF 246 were selected as candidate antagonists in the second screening step. a. Traces of typical current responses to PF 419 (30 µM) and BaCl2 (2 mM) in the oocyte expressing the GIRK1/4 channel. The striped and filled bars represent the duration of the application of PF 419 and BaCl2, respectively. b. The normalized current responses to the selected PF compounds by the response to BaCl2.
Concentration-Response Relationships of the Identified Agonist and Antagonists to GIRK Channels. a. Current response was normalized by the response to ethanol (100 mM). b-d. Current responses were normalized by the response to BaCl2 (2 mM). *P < 0.005 between GIRK1/2 and GIRK1/4. **P < 0.001 between GIRK1/2 and GIRK1/4.
G protein-activated inwardly rectifying K(+) (GIRK) channels have been known to play a key role in the rewarding and analgesic effects of opioids. To identify potent agonists and antagonists to GIRK channels, we examined various compounds for their ability to activate or inhibit GIRK channels. A total of 503 possible compounds with low molecular weight were selected from a list of fluoxetine derivatives at Pfizer Japan Inc. We screened these compounds by a Xenopus oocyte expression system. GIRK1/2 and GIRK1/4 heteromeric channels were expressed on Xenopus laevis oocytes at Stage V or VI. A mouse IRK2 channel, which is another member of inwardly rectifying potassium channels with similarity to GIRK channels, was expressed on the oocytes to examine the selectivity of the identified compounds to GIRK channels. For electrophysiological analyses, a two-electrode voltage clamp method was used. Among the 503 compounds tested, one compound and three compounds were identified as the most effective agonist and antagonists, respectively. All of these compounds induced only negligible current responses in the oocytes expressing the IRK2 channel, suggesting that these compounds were selective to GIRK channels. These effective and GIRK-selective compounds may be useful possible therapeutics for drug dependence and pain.
The protease-activated receptors (PARs) play a pivotal role in inflammatory and nociceptive processes. PARs have raised considerable interest because of their capacity to regulate numerous aspects of viscera physiology and pathophysiology. The present article summarizes research on PARs and proteases as signalling molecules in visceral pain. In particular, experiments in animal models suggest that PAR2 is important for visceral hypersensitivity. Moreover, endogenous PAR2 agonists seem to be released by colonic tissue of patients suffering from irritable bowel syndrome, suggesting a role for this receptor in visceral pain perception. Thus, PARs, together with proteases that activate them, represent exciting targets for therapeutic intervention on visceral pain.
Inhibitory effects of PCP on GIRK channels expressed in Xenopus oocytes. (A) Top, in an oocyte injected with GIRK1 and GIRK2 mRNA, current responses to 10 µM and 100 µM PCP and to 3 mM Ba2+, a Kir channel blocker. Middle, in an oocyte injected with Kir1.1 mRNA, current responses to 100 µM PCP and to 3 mM Ba2+. Bottom, in an uninjected oocyte, no significant current responses to 100 µM PCP or 3 mM Ba2+. Asterisks show the zero current level. Bars show the duration of application. (B) Concentration-dependent inhibition of GIRK channels by PCP. The magnitudes of inhibition of GIRK currents by PCP were compared with the current components sensitive to 3 mM Ba2+.
Comparison of the effects of five addictive drugs: PCP, cocaine, methylphenidate (MPH), methamphetamine (MAP) and MDMA, on GIRK channels. Drug concentration was 100 µM. The magnitudes of inhibition of GIRK currents by the drugs were compared with the 3 mM Ba2+-sensitive current components. Data except for PCP are from our previous study [12].
Addictive drugs, such as opioids, ethanol, cocaine, amphetamine, and phencyclidine (PCP), affect many functions of the nervous system and peripheral organs, resulting in severe health problems. G protein-activated inwardly rectifying K(+) (GIRK, Kir3) channels play an important role in regulating neuronal excitability through activation of various Gi/o protein-coupled receptors including opioid and CB(1) cannabinoid receptors. Furthermore, the channels are directly activated by ethanol and inhibited by cocaine at toxic levels, but not affected by methylphenidate, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) at toxic levels. The primary pharmacological action of PCP is blockade of N-methyl-D-aspartate (NMDA) receptor channels that are associated with its psychotomimetic effects. PCP also interacts with several receptors and channels at relatively high concentrations. However, the molecular mechanisms underlying the various effects of PCP remain to be clarified. Here, we investigated the effects of PCP on GIRK channels using the Xenopus oocyte expression system. PCP weakly but significantly inhibited GIRK channels at micromolar concentrations, but not Kir1.1 and Kir2.1 channels. The PCP concentrations effective in inhibiting GIRK channels overlap clinically relevant brain concentrations in severe intoxication. The results suggest that partial inhibition of GIRK channels by PCP may contribute to some of the toxic effects after overdose.
(A). Schematic of signal transduction in (i) a normal neuron in a healthy brain, versus (ii) a damaged demyelinated neuron in a
patient affected by MS. In MS the immune system attacks the white matter of the brain in a combined insult involving auto-reactive T cells,
B cells, macrophages and activated microglia. Inflammation leads to the formation of plaques, followed by the destruction of the protective
myelin sheath. Myelin damage leads to impaired signal transduction or blockage, resulting in the clinical symptoms of MS. Removal or damage
of the myelin sheath leaves the nerve axon exposed and subject to direct injury [24, 29, 31, 43]. (B). The primary sources of ROS and the
cellular occurrences that may lead to oligodendrocyte and neuronal loss in EAE and MS. Modified from ref. [24]. (C). Autoimmune damage
causes the supportive astrocytes to be lost or damaged, and repopulation by activated microglia follows. Activated microglia release glutamate,
further increasing glutamate levels, which in turn promotes excitotoxic neuronal death by excessive NMDA receptor activation. The
activation of T-cells specific against CNS-derived antigens follows. T-cells penetrate the BBB and release further glutamate [29, 39, 40, 45].
APC= Antigen presenting cell. (D). Possible effects of BARF1 protein on cells of the CNS and proposed mechanisms based on previous
observations in a variety of cell types.
Multiple sclerosis and neurodegenerative diseases in which cells of the central nervous system (CNS) are lost or damaged are rapidly increasing in frequency, and there is neither effective treatment nor cure to impede or arrest their destructive course. The Epstein-Barr virus is a human gamma-herpesvirus that infects more than 90% of the human population worldwide and persisting for the lifetime of the host. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. Individuals with a history of symptomatic primary EBV infection, called infectious mononucleosis, carry a moderately higher risk of developing multiple sclerosis (MS). It is not known how EBV infection potentially promotes autoimmunity and central nervous system (CNS) tissue damage in MS. Recently it has been found that EBV isolates from different geographic regions have highly conserved BARF1 epitopes. BARF1 protein has the neuroprotective and mitogenic activity, thus may be useful to combat and overcome neurodegenerative disease. BARF1 protein therapy can potentially be used to enhance the neuroprotective activities by combinational treatment with anti-inflammatory antagonists and neuroprotectors in neural disorders.
Effects of Cannabinoid Receptor Agonists in Healthy Volunteers Submitted to Acute Noxious Stimuli
Cannabis extracts and synthetic cannabinoids are still widely considered illegal substances. Preclinical and clinical studies have suggested that they may result useful to treat diverse diseases, including those related with acute or chronic pain. The discovery of cannabinoid receptors, their endogenous ligands, and the machinery for the synthesis, transport, and degradation of these retrograde messengers, has equipped us with neurochemical tools for novel drug design. Agonist-activated cannabinoid receptors, modulate nociceptive thresholds, inhibit release of pro-inflammatory molecules, and display synergistic effects with other systems that influence analgesia, especially the endogenous opioid system. Cannabinoid receptor agonists have shown therapeutic value against inflammatory and neuropathic pains, conditions that are often refractory to therapy. Although the psychoactive effects of these substances have limited clinical progress to study cannabinoid actions in pain mechanisms, preclinical research is progressing rapidly. For example, CB(1)mediated suppression of mast cell activation responses, CB(2)-mediated indirect stimulation of opioid receptors located in primary afferent pathways, and the discovery of inhibitors for either the transporters or the enzymes degrading endocannabinoids, are recent findings that suggest new therapeutic approaches to avoid central nervous system side effects. In this review, we will examine promising indications of cannabinoid receptor agonists to alleviate acute and chronic pain episodes. Recently, Cannabis sativa extracts, containing known doses of tetrahydrocannabinol and cannabidiol, have granted approval in Canada for the relief of neuropathic pain in multiple sclerosis. Further double-blind placebo-controlled clinical trials are needed to evaluate the potential therapeutic effectiveness of various cannabinoid agonists-based medications for controlling different types of pain.
Effects of Adenosine on Cerebral Ischemia: Studies with Transgenic Animals
Adenosine modulates peripheral immune cell function. Adenosine modulates the activity of peripheral immune cells differentially depending on the receptors that are activated. Overall, activation of A 2A and A 2B receptors directs immune cells to an antiinflammatory response. This property could be harnessed in stroke to reduce inflammatory damage in the CNS caused by infiltrating activated immune cells. Stimulation via A 1 receptors on immune cells tends to increase migration and activity, but also leads to angiogenesis through their interaction with cerebral vascular endothelial cells.  
Peripheral immune involvement following ischemic brain injury. Neurons and astrocytes damaged by ischemia release proinflammatory factors such as TNF-α into the brain parenchyma. These pro-inflammatory molecules in turn diffuse and bind receptors on vascular endothelial cells. Activation of endothelial cells via TNF and related receptors leads to nuclear factor kappa B (NFκB) activation and nuclear translocation. In the nucleus, NFκB activity induces transcription of adhesion molecules and chemokines, including E-selectin, P-selectin, vascular cell adhesion molecule (VCAM1), intercellular adhesion molecule (ICAM1) and heparin sulfate proteoglycan. Peripheral immune cells traveling through the vasculature express receptors to adhesion molecules (i.e. PSGL-1 for selectins, α4-βintegrin for VCAM1). As they survey the body, leukocytes roll along the activated endothelium, adhere via adhesion molecules and become activated themselves, then extravasate into the brain parenchyma.  
Stroke is a leading cause of morbidity and mortality in the United States. Despite intensive research into the development of treatments that lessen the severity of cerebrovascular injury, no major therapies exist. Though the potential use of adenosine as a neuroprotective agent in the context of stroke has long been realized, there are currently no adenosine-based therapies for the treatment of cerebral ischemia and reperfusion. One of the major obstacles to developing adenosine-based therapies for the treatment of stroke is the prevalence of functional adenosine receptors outside the central nervous system. The activities of peripheral immune and vascular endothelial cells are particularly vulnerable to modulation via adenosine receptors. Many of the pathophysiological processes in stroke are a direct result of peripheral immune infiltration into the brain. Ischemic preconditioning, which can be induced by a number of stimuli, has emerged as a promising area of focus in the development of stroke therapeutics. Reprogramming of the brain and immune responses to adenosine signaling may be an underlying principle of tolerance to cerebral ischemia. Insight into the role of adenosine in various preconditioning paradigms may lead to new uses for adenosine as both an acute and prophylactic neuroprotectant.
Two strategies for TMS. (A) Conventional procedures comprise adaptor tagging of fragmented genomic DNAs (Steps 1 and 2), target enrichment by hybridization (Steps 3 and 4) and bisulfite treatment of enriched library DNAs (Step 5) followed by PCR amplification (Step 6). The bisulfite treatment (Step 5) induces DNA breaks, inevitably leading to severe loss of intact sequencing template molecules. (B) PBAT-mediated procedure comprises target enrichment by hybridization (Steps 1-3), bisulfite treatment (Step 4) and adaptor tagging (Step 5), thereby circumventing the bisulfite-induced loss of intact sequencing template molecules.
Performance of PBAT-mediated TMS. (A) Target coverage. The fraction of the targets covered by differing minimal depth of reads was shown for the six PBAT and one Methyl-Seq libraries generated from the indicated amount of input DNA. Note that the average read depth of the Methyl-Seq library was twice or more higher than those of the PBAT libraries (Table 1). (B) Consistency among TMS data. Methylation levels were compared among the six TMS libraries generated from 3,000 to 10 ng of human genomic DNA using the PBAT-mediated procedure as well as the one generated from 3,000 ng of input DNA using the original Methyl-Seq protocol (DRA002274-002280). The numbers and the images in the boxes above and below the diagonal indicated the coefficients of determination (R 2 ) and the scatter plot of methylation levels, respectively, between all the possible combinations among the seven data sets. The moving averages of methylation levels (window size, 500 bp; step size, 250 bp) were calculated based on CpGs covered by 20 or more reads. (C) A snapshot of TMS data. Data around the imprinted control region (ICR) for PEG3 were compared among the seven libraries generated with either PBAT or Methyl-Seq using the indicated amount of input DNA. Red bars and grey shadows indicated the methylation levels of individual CpG sites and the depth of reads, respectively. Note that most reads were mapped to the bottom strand, as the RNA probes used in the experiment were designed from the top strand. As expected, a region around the PEG3 promoter showed ∼50% methylation level due to the imprinted monoallelic methylation. The green dashed box denoted the ICR of PEG3 (chr.19: 57,351,728 to 57,352,173 in hg19
Consistency between TMS and WGBS data. Methylation levels were compared between the TMS data (DRA002281) (Table 1) and a publicly available WGBS data (DRA002248) on human IMR90 cells using the CpG sites covered by 20 or more reads. Methylation levels were plotted for moving windows (window size, 500 bp; stepping size, 250 bp) (A) and for individual CpG sites (B). 
The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.
Addiction involves complex physiological processes, and is characterised not only by broad phenotypic and behavioural traits, but also by ongoing molecular and cellular adaptations. In recent years, increasingly effective and novel techniques have been developed to unravel the molecular implications of addiction. Increasing evidence has supported a contribution of the nuclear transcription factor CREB in the development of addiction, both in contribution to phenotype and expression in brain regions critical to various aspects of drug-seeking behaviour and drug reward. Abstracting from this, models have exploited these data by removing the CREB gene from the developing or developed mouse, to crucially determine its impact upon addiction-related processes. More recent models, however, hold greater promise in unveiling the contribution of CREB to disorders such as addiction.
Time (in sec) spent in each compartment (black with smooth floor or white with rough floor) in the Pre-Conditioning Phase, for trained and untrained groups. Results shown are mean ± SEM; n=6-10 rats per group.
Time (in sec) spent in compartments paired with saline or amphetamine (2 after conditioning, for trained and untrained groups. Results shown are mean ± SEM; n=6-10 rats per group. # p < 0.05; ## p < 0.01.
Amphetamines exert their persistent addictive effects by activating brain's reward pathways, perhaps through the release of dopamine in the nucleus accumbens (and/or in other places). On the other hand, there is a relationship between dopamine and all behavioural aspects that involve motor activity and it has been demonstrated that exercise leads to an increase in the synthesis and release of dopamine, stimulates neuroplasticity and promotes feelings of well-being. Moreover, exercise and drugs of abuse activate overlapping neural systems. Thus, our aim was to study the influence of chronic exercise in the mechanism of addiction using an amphetamine-induced conditioned-place-preference in rats. Adult male Sprague-Dawley rats were randomly separated in groups with and without chronic exercise. Chronic exercise consisted in a 8 week treadmill running program, with increasing intensity. The conditioned place preference test was performed in both groups using a procedure and apparatus previously established. A 2 amphetamine or saline solution was administered intraperitonially according to the schedule of the conditioned place preference. Before conditioning none of the animals showed preference for a specific compartment of the apparatus. The used amphetamine dose in the conditioning phase was able to produce a marked preference towards the drug-associated compartment in the group without exercise. In the animals with exercise a significant preference by the compartment associated with saline was observed. These results lead us to conclude that a previous practice of regular physical activity may help preventing amphetamine addiction in the conditions used in this test.
There are an estimated 11.7 million methamphetamine (MA) abusers in the United States and epidemics of MA addiction are occurring worldwide. In our human laboratory and outpatient clinical trials we use innovative methods to quantify the severity of MA addiction and test biomarkers that may predict response to therapy or risk of relapse. One potential biomarker of addiction is the quantity of abused drug intake. Qualitative urinalysis is used in clinical trials and during treatment but provides only a binary outcome measure of abuse. Using non-pharmacologic doses of deuterium labeled l-MA we have developed a continuous quantitative measure to estimate the bioavailable amount of MA addicts ingest. Brain Derived Neurotrophic Factor is a neurotrophin that encourages growth and differentiation of new neurons and synapses. Low BDNF levels are seen in many addictive disorders and BDNF is elevated in recovering MA addicts, suggesting BDNF may be a marker of MA addiction. We are investigating the effects of controlled doses of MA on BDNF levels and gene regulation and measuring BDNF in our clinical trials. We believe both patients and clinical researches will benefit from the addition of new, objective and quantifiable outcome measures that reflect disease severity and recovery from addiction.
IDARS is an acronym for the International Drug Abuse Research Society. Apart from our scientific and educational purposes, we communicate information to the general and scientific community about substance abuse and addiction science and treatment potential. Members of IDARS are research scientists and clinicians from around the world, with scheduled meetings across the globe. IDARS is developing a vibrant and exciting international mechanism not only for scientific interactions in the domain of addiction between countries but also ultimately as a resource for informing public policy across nations. Nonetheless, a lot more research needs to be done to better understand the neurobiological basis of drug addiction - A challenge for IDARS scientists.
The glutamatergic synapse and pharmacological targets for increasing synaptic plasticity and extinction learning. NMDA receptor function can by potentiated by activation of the glycine co-agonist binding site with the partial NMDA receptor agonist DCS or D-serine. Alternatively, NMDA receptor function can be also increased indirectly by inhibition of GlyT1 function, which elevates extracellular levels of the NMDA receptor co-agonist glycine, or by positive allosteric modulation of mGluR5, which indirectly increase NMDA receptor function. AMPA receptor activity can be enhanced with AMPA receptor potentiators. Finally, a nonspecific increase in extracellular glutamate can be obtained by stimulation of the glial cystine-glutamate exchanger (xc) with cystine derived from cystine prodrugs such as N-acetylcysteine.
Chemical structures of various systemically active pharmacological agents, categorized by mechanism of action, that may increase extinction learning via subtle enhancement of glutamatergic transmission.
The persistence of the motivational salience of drug-related environmental cues and contexts is one of the most problematic obstacles to successful treatment of drug addiction. Behavioral approaches to extinguishing the salience of drug-associated cues, such as cue exposure therapy, have generally produced disappointing results which have been attributed to, among other things, the context specificity of extinction and inadequate consolidation of extinction learning. Extinction of any behavior or conditioned response is a process of new and active learning, and increasing evidence suggests that glutamatergic neurotransmission, a key component of the neural plasticity that underlies normal learning and memory, is also involved in extinction learning. This review will summarize findings from both animal and human studies that suggest that pharmacological enhancement of glutamatergic neurotransmission facilitates extinction learning in the context of drug addiction. Pharmacological agents that have shown potential efficacy include NMDA partial agonists, mGluR5 receptor positive allosteric modulators, inhibitors of the GlyT1 glycine transporter, AMPA receptor potentiators, and activators of the cystine-glutamate exchanger. These classes of cognition-enhancing compounds could potentially serve as novel pharmacological adjuncts to cue exposure therapy to increase success rates in attenuating cue-induced drug craving and relapse.
A schematic model of somatic state activation and decision-making. (a) The amygdala is a trigger structure for emotional (somatic) states from primary inducers. It couples the features of primary inducers, which can be processed subliminally (e.g., via the thalamus) or explicitly (e.g., via early sensory and high-order association cortices), with effector structures that trigger the emotional/somatic response. (b) The ventromedial prefrontal (VM) cortex is a trigger structure for emotional (somatic) states from secondary inducers. It couples knowledge of events held temporarily in working memory (which is dependent on dorsolateral prefrontal (DLF) cortices) to effector structures that induce the somatic responses, and to structures holding representations of previous feeling states (e.g., Insula and Somatosensory I (SI) and Somatosensory II (SII) cortices).  
A diagram illustrating three different levels at which somatic states can bias decisions v i a the release of neurotransmitters (NT). (1) Dopamine biases decisions covertly (perhaps through action in the striatum and affective sector of anterior cingulate (Brodmann Area (BA) 25 and lower 24, 32). (2) Serotonin biases decisions overtly (perhaps through action in the cognitive sector of anterior cingulate and probably the adjacent SMA (Supplementary Motor Area)). (3) Somatic states also bias working memory in the LOF (lateral orbitofrontal and dorsolateral regions of the prefrontal cortex); They help endorse or reject " thoughts " , " options " , or " scenarios " brought to mind during the pondering of decisions, i.e., before their translation into action. The neurotransmitter system that mediates this biasing function remains to be determined.  
Similar to patients with orbitofrontal cortex lesions, substance dependent individuals (SDI) show signs of impairments in decision-making, characterised by a tendency to choose the immediate reward at the expense of severe negative future consequences. The somatic-marker hypothesis proposes that decision-making depends in many important ways on neural substrates that regulate homeostasis, emotion and feeling. According to this model, there should be a link between abnormalities in experiencing emotions in SDI, and their severe impairments in decision-making in real-life. Growing evidence from neuroscientific studies suggests that core aspects of substance addiction may be explained in terms of abnormal emotional guidance of decision-making. Behavioural studies have revealed emotional processing and decision-making deficits in SDI. Combined neuropsychological and physiological assessment has demonstrated that the poorer decision-making of SDI is associated with altered reactions to reward and punishing events. Imaging studies have shown that impaired decision-making in addiction is associated with abnormal functioning of a distributed neural network critical for the processing of emotional information, including the ventromedial cortex, the amygdala, the striatum, the anterior cingulate cortex, and the insular/somato-sensory cortices, as well as non-specific neurotransmitter systems that modulate activities of neural processes involved in decision-making. The aim of this paper is to review this growing evidence, and to examine the extent of which these studies support a somatic-marker model of addiction.
Economically important traits in many species generally show polygenic, quantitative inheritance. The components of genetic variation (additive, dominant and epistatic effects) of these traits conferred by multiple genes in shared biological pathways remain to be defined. Here, we investigated 11 full-length genes in cellulose biosynthesis, on 10 growth and wood-property traits, within a population of 460 unrelated Populus tomentosa individuals, via multi-gene association. To validate positive associations, we conducted single-marker analysis in a linkage population of 1,200 individuals. We identified 118, 121, and 43 associations (P< 0.01) corresponding to additive, dominant, and epistatic effects, respectively, with low to moderate proportions of phenotypic variance (R(2)). Epistatic interaction models uncovered a combination of three non-synonymous sites from three unique genes, representing a significant epistasis for diameter at breast height and stem volume. Single-marker analysis validated 61 associations (false discovery rate, Q ≤ 0.10), representing 38 SNPs from nine genes, and its average effect (R(2) = 3.8%) nearly 2-fold higher than that identified with multi-gene association, suggesting that multi-gene association can capture smaller individual variants. Moreover, a structural gene-gene network based on tissue-specific transcript abundances provides a better understanding of the multi-gene pathway affecting tree growth and lignocellulose biosynthesis. Our study highlights the importance of pathway-based multiple gene associations to uncover the nature of genetic variance for quantitative traits and may drive novel progress in molecular breeding. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
Adenosine is produced primarily by the metabolism of ATP and mediates its physiological actions by interacting primarily with adenosine receptors (ARs) on the plasma membranes of different cell types in the body. Activation of these G protein-coupled receptors promotes activation of diverse cellular signaling pathways that define their tissue-specific functions. One of the major actions of adenosine is cytoprotection, mediated primarily via two ARs - A(1) (A(1)AR) and A(3) (A(3)AR). These ARs protect cells exposed to oxidative stress and are also regulated by oxidative stress. Stress-mediated regulation of ARs involves two prominent transcription factors - activator protein-1 (AP-1) and nuclear factor (NF)-κB - that mediate the induction of genes important in cell survival. Mice that are genetically deficient in the p50 subunit of NF-κB (i.e., p50 knock-out mice) exhibit altered expression of A(1)AR and A(2A)AR and demonstrate distinct behavioral phenotypes under normal conditions or after drug challenges. These effects suggest an important role for NF-κB in dictating the level of expression of ARs in vivo, in regulating the cellular responses to stress, and in modifying behavior.
Possible mechanisms for activity-dependent adenosine release. 1) ATP is released by exocytosis (either from a neuron or glial cell) and subsequently metabolised in the extracellular space to produce adenosine. 2) An unspecified interposed transmitter (?) is released by exocytosis to act on a downstream cell causing the release of adenosine by an unknown mechanism (?). 3) Direct exocytotic release of vesicular adenosine. 
Presynaptic inhibition by endogenous adenosine is increased during high frequency stimulation at the Calyx of Held Excitatory postsynaptic currents (EPSCs) were recorded from MNTB principal neurons in P5-7 rats in response to stimulation of input fibres. A) Averaged EPSCs during a train of 30 stimuli at 10 Hz. The first EPSC (I 0 ) and the 27th-30th EPSCs, before (black) and during (red) application of CPT (0.5 M), are shown (superimposed). B) Synaptic depression during 10 Hz stimulation. EPSCs during a train are normalized in amplitude to the first EPSC. Mean amplitudes and S.E.M.s of EPSCs (from five cells with significant increase in mean amplitude of 20th-30th EPSCs (I ss ) after CPT application, D) during 10 Hz stimulation are plotted, before ( ) and during ( ) CPT application. C, Time plot of I 0 and I ss in a cell. Bath application of CPT (bar) increased I ss with no effect on I 0. Mean amplitude of I ss before CPT application is indicated by a dashed line. D) mean amplitudes of I ss before and after application of CPT in seven cells. Difference was statistically significant (*P < 0.05) in five cells shown in B, but insignificant in two other cells. Taken with permission from [33]. 
Adenosine is perhaps the most important and universal modulator in the brain. The current consensus is that it is primarily produced in the extracellular space from the breakdown of previously released ATP. It is also accepted that it can be released directly, as adenosine, during pathological events primarily by equilibrative transport. Nevertheless, there is a growing realization that adenosine can be rapidly released from the nervous system in a manner that is dependent upon the activity of neurons. We consider three competing classes of mechanism that could explain neuronal activity dependent adenosine release (exocytosis of ATP followed by extracellular conversion to adenosine; exocytotic release of an unspecified transmitter followed by direct non-exocytotic adenosine release from an interposed cell; and direct exocytotic release of adenosine) and outline discriminatory experimental tests to decide between them. We review several examples of activity dependent adenosine release and explore their underlying mechanisms where these are known. We discuss the limits of current experimental techniques in definitively discriminating between the competing models of release, and identify key areas where technologies need to advance to enable definitive discriminatory tests. Nevertheless, within the current limits, we conclude that there is evidence for a mechanism that strongly resembles direct exocytosis of adenosine underlying at least some examples of neuronal activity dependent adenosine release.
Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions.
Conditions that Increase Adenosine in the CNS
The metabolic relationship between ketones and adenosine. Compounds upregulated by a ketogenic diet or exogenous ketones are italicized. (1) During ketolytic metabolism, the ketone bodies β-hydroxybutyrate (and its breakdown products acetone and acetoacetate) are either generated locally or hepatically and transported via the blood to other tissues (such as brain). Ketones are converted intracellularly into acetyl-CoA which enters the tricarboxylic acid cycle. (2) This mitochondrial energy cycle generates, at multiple steps (----), protons and electrons that are channeled to the electron transport chain by NADH and FADH2 (β-hydroxybutyrate conversion to acetoacetate also contributes). Many steps of the tricarboxylic acid cycle are omitted for simplicity. (3) The electron transport chain drives an electrochemical gradient across the mitochondrial outer membrane and ultimately oxidative phosphorylation which forms ATP from ADP and phosphate (Pi) by ATP synthase. (4) Enhanced ATP can be converted to phosphocreatine for energy storage, or broken down to its core molecule, adenosine. Adenosine levels inside and outside of the cell membrane are influenced concurrently by an equilibrative transporter. Due to the very large ATP / adenosine ratio inside the cell, a small increase in intracellular ATP could translate into a large relative increase in intracellular, and thus extracellular, adenosine.
For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a "retaliatory metabolite." As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor-based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed.
Top-cited authors
Bayani Uttara
Raghunath Totaram Mahajan
  • Post Graduate College of Science, Technology and Research, Jalagaon
Ajay vikram Singh
  • The German Federal Institute for Risk Assessment (BfR)
Paolo Zamboni
  • University of Ferrara
Danijela Krstic
  • University of Belgrade