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A sensitive HPLC method using fluorescence detection was developed to determine kynurenic acid (KYNA) level in rat cerebrospinal fluid (CSF). The method development was accomplished by screening different columns, optimizing zinc acetate concentration and determining the optimal HPLC flow rate. This method allowed direct injection of the CSF samples onto an Xselect C18 column and KYNA levels were measured fluorometrically by forming a fluorescent complex with zinc acetate that was delivered post-column. The limit of quantitation was 0.2 n m with 30 μL injection, corresponding to 6 fmol (signal-to-noise ratio = 10). The improved sensitivity enabled the measurement of KYNA in naive and drug-treated rat CSF.

Acetyl-l-carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a high-throughput scale, a new and simple HILIC-MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via ‘dilute and shoot’ directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC-MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high-throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies. Copyright © 2015 John Wiley & Sons, Ltd.

RATIONALE: Research on disorders of the central nervous system (CNS) has shown that an imbalance in the levels of specific endogenous neurotransmitters may underlie certain CNS diseases. These alterations in neurotransmitter levels may provide insight into pathophysiology, but can also serve as disease and pharmacodynamic biomarkers. To measure these potential biomarkers in vivo, the relevant sample matrix is cerebrospinal fluid (CSF), which is in equilibrium with the brain's interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Accurate analysis of these potential biomarkers can be challenging due to low CSF sample volume, low analyte levels, and potential interferences from other endogenous compounds. METHODS: A protocol has been established for effective method development of bioanalytical assays for endogenous compounds in CSF. Database searches and standard-addition experiments are employed to qualify sample preparation and specificity of the detection thus evaluating accuracy and precision. RESULTS: This protocol was applied to the study of the histaminergic neurotransmitter system and the analysis of histamine and its metabolite 1-methylhistamine in rat CSF. CONCLUSIONS: The protocol resulted in a specific and sensitive novel method utilizing pre-column derivatization ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS), which is also capable of separating an endogenous interfering compound, identified as taurine, from the analytes of interest. Copyright © 2013 John Wiley & Sons, Ltd. An imbalance in the levels of specific endogenous neurotransmitters has been implicated in the etiology of many disorders of the central nervous system. For example, reduced levels of monoamines have been suggested to play a role in major depressive disorders, [1] and aberrant levels of dopamine and glutamate have been hypothesized to underlie schizophrenia. [2] It is therefore not surprising that many of the medications developed to treat these disorders target mechanisms intended to restore neurotransmitter balance in an effort to ameliorate disease symptoms. In preclinical research, neurotransmitter levels can be measured either in isolated brain tissue from terminal experiments, or in vivo using microdialysis to quantify the levels of these molecules in awake and freely moving animals. However, these methods are not applicable to the clinical setting, and thus there is an interest in developing methods to detect neurotransmitters and their metabolites as potential biomarkers in the cerebrospinal fluid (CSF) of animals and humans, to enable translation of preclinical

DeltaFosB is a Fos family transcription factor that is induced by chronic exposure to cocaine and other drugs of abuse in the nucleus accumbens and related striatal regions, brain regions that are important for the behavioral effects of these drugs. To better understand the mechanisms by which DeltaFosB contributes to the effects of chronic drug treatment, we used DNA microarray analysis to identify genes that are regulated in the nucleus accumbens upon DeltaFosB expression in inducible bitransgenic mice. One of the most highly regulated genes was that encoding a subunit of another transcription factor, nuclear factor-kappaB (NF-kappaB). Subsequent experiments confirmed the induction of NF-kappaB in the nucleus accumbens of mice overexpressing DeltaFosB as well as in wild-type mice treated chronically, but not acutely, with cocaine. These results establish NF-kappaB as a putative target for DeltaFosB and implicate NF-kappaB signaling pathways in the long-term adaptations of nucleus accumbens neurons to cocaine.

Herein we describe a novel series of compounds from which varenicline (1, 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine) has been identified for smoking cessation. Neuronal nicotinic acetylcholine receptors (nAChRs) mediate the dependence-producing effects of nicotine. We have pursued alpha4beta2 nicotinic receptor partial agonists to inhibit dopaminergic activation produced by smoking while simultaneously providing relief from the craving and withdrawal syndrome that accompanies cessation attempts. Varenicline displays high alpha4beta2 nAChR affinity and the desired in vivo dopaminergic profile.

The long lasting antidepressant response seen following acute, i.v. ketamine administration in patients with treatment-resistant depression (TRD) is thought to result from enhanced synaptic plasticity in cortical and hippocampal circuits. Using extracellular field recordings in rat hippocampal slices, we show that a single dose of the non-selective NMDA receptor antagonist ketamine or CP-101,606, a selective antagonist of the NR2B subunit of the NMDA receptor, enhances hippocampal synaptic plasticity induced with high frequency stimulation (HFS) 24h after dosing - a time at which plasma concentrations of the drug are no longer detectable in the animal. These results indicate that acute inhibition of NMDA receptors containing the NR2B subunit can lead to long-lasting changes in hippocampal plasticity.

Acetyl-L-carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a high-throughput scale, a new and simple HILIC-MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via 'dilute and shoot' directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC-MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high-throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies.

One result of globalization is that more of what people consume comes from distant regions through complex transactions hidden from ordinary view. To illuminate such flows, scholars often trace commodity chains following one product from inception to sale. Although most of us cannot escape being involved in commodity chains, we can participate with greater or less responsibility. This article outlines the advantages of studying the coffee chain to understand its relationship to consumer choice and environmental issues.

The effect of nicotine on the major human neuronal nicotinic receptor (alpha 4 beta 2 subtype) was studied in permanently transfected HEK 293 cells. Prolonged exposure to low concentrations of nicotine (1 microM) increased epibatidine binding but functionally deactivated the nicotinic receptor, abolishing Ca2+ influx in response to an acute nicotine challenge. Deactivation could also be caused by down-regulating protein kinase C (PKC) activity with 0.5 microM phorbol-12,13-dibutyrate or briefly incubating cells with the PKC inhibitor NPC-15437. Recovery from receptor deactivation caused by either nicotine treatment or PKC inhibition occurred slowly (4-6 hr). Reversal of nicotine-induced deactivation was accelerated by the addition of inhibitors of protein phosphatases 2A and 2B. These data suggest a hypothetical mechanism of nicotine-induced deactivation that involves dephosphorylation of nicotinic receptors at PKC phosphorylation sites.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Research on disorders of the central nervous system (CNS) has shown that an imbalance in the levels of specific endogenous neurotransmitters may underlie certain CNS diseases. These alterations in neurotransmitter levels may provide insight into pathophysiology, but can also serve as disease and pharmacodynamic biomarkers. To measure these potential biomarkers in vivo, the relevant sample matrix is cerebrospinal fluid (CSF), which is in equilibrium with the brain's interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Accurate analysis of these potential biomarkers can be challenging due to low CSF sample volume, low analyte levels, and potential interferences from other endogenous compounds.A protocol has been established for effective method development of bioanalytical assays for endogenous compounds in CSF. Database searches and standard-addition experiments are employed to qualify sample preparation and specificity of the detection thus evaluating accuracy and precision.This protocol was applied to the study of the histaminergic neurotransmitter system and the analysis of histamine and its metabolite 1-methylhistamine in rat CSF.The protocol resulted in a specific and sensitive novel method utilizing pre-column derivatization ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS), which is also capable of separating an endogenous interfering compound, identified as taurine, from the analytes of interest.

As the main excitatory neurotransmitter in the mammalian central nervous system, glutamate is critically involved in most aspects of CNS function. Given this critical role, it is not surprising that glutamatergic dysfunction is associated with many CNS disorders. In this chapter, we review the literature that links aberrant glutamate neurotransmission with CNS pathology, with a focus on neurodegenerative diseases. The biology and pharmacology of the various glutamate receptor families are discussed, along with data which links these receptors with neurodegenerative conditions. In addition, we review progress that has been made in developing small molecule modulators of glutamate receptors and transporters, and describe how these compounds have helped us understand the complex pharmacology of glutamate in normal CNS function, as well as their potential for the treatment of neurodegenerative diseases.

The type 1 glycine transporter plays an important in regulating homeostatic glycine levels in the brain that are relevant to the activation of the NMDA receptor by the excitatory neurotransmitter glutamate. We describe herein the structure-activity relationships (SAR) of a structurally novel class of GlyT1 inhibitors following on a lead derived from high throughput screening, which shows good selectivity for GlyT1 and potent activity in elevating CSF levels of glycine.

Casein kinase Iepsilon (CKIepsilon) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC(50) = 7.7 +/- 2.2 nM) and selective (30-fold with respect to 42 additional kinases) inhibitor of CKIepsilon in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (-1.97 +/- 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose- and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these drug-induced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t(1/2) = 0.46 +/- 0.04 h). Together, these results suggest that inhibition of CKIepsilon yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.

The preclinical pharmacology of the alpha4beta2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline, a novel smoking cessation agent is described. Varenicline binds with subnanomolar affinity only to alpha4beta2 nAChRs and in vitro functional patch clamp studies in HEK cells expressing nAChRs show that varenicline is a partial agonist with 45% of nicotine's maximal efficacy at alpha4beta2 nAChRs. In neurochemical models varenicline has significantly lower (40-60%) efficacy than nicotine in stimulating [(3)H]-dopamine release from rat brain slices in vitro and in increasing dopamine release from rat nucleus accumbens in vivo, while it is more potent than nicotine. In addition, when combined with nicotine, varenicline effectively attenuates the nicotine-induced dopamine release to the level of the effect of varenicline alone, consistent with partial agonism. Finally, varenicline reduces nicotine self-administration in rats and supports lower self-administration break points than nicotine. These data suggest that varenicline can reproduce to some extent the subjective effects of smoking by partially activating alpha4beta2 nAChRs, while preventing full activation of these receptors by nicotine. Based on these findings, varenicline was advanced into clinical development and recently shown to be an effective and safe aid for smoking cessation treatment.

Two single-chain Fv (sFv) antibodies (C8 and H2) specific for Mch3/caspase-7, a component in the signaling pathway for induction of apoptosis, were genetically fused to different intracellular targeting signals and analyzed by expression in mammalian cells. Immunofluorescence microscopy confirmed that these anti-caspase-7 intrabodies were expressed in the cellular compartments dictated by their C-terminal trafficking signals. Cytosolic caspase-7 was successfully retargeted to different subcellular compartments by specific intrabodies through direct association of antigen with intrabody. Sequestration of caspase-7 in nuclei had a significant biological impact in that the expression of a nuclear-targeted anti-caspase-7 intrabody in a stable Jurkat cell line markedly inhibited staurosporine-induced apoptosis. The criteria for choosing an optimal intrabody were also evaluated in this study. A gene dosage titration study demonstrated that the C8 intrabody was more potent in retargeting of caspase-7 than the H2 intrabody, even though the H2 sFv had a higher affinity for caspase-7 than the C8. Pulse-chase experiments and western blot analysis revealed that the anti-caspase-7 C8 sFv intrabodies exhibited a long half-life (>8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.

Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.

We have investigated the possible involvement of the synaptic vesicle protein synapsin IIa in synapse development. Synapsin IIa was introduced into Xenopus embryonic spinal neurons by early blastomere injection, and nerve-muscle cultures were prepared. Synaptic currents were measured by comparing synapses in which the presynaptic neuron either contained [syn IIa (+)] or lacked (control) exogenous synapsin IIa. Syn IIa (+) synapses had a 3.6-fold increase in the frequency and a 2.1-fold increase in the amplitude of spontaneous synaptic currents, compared to controls, after 2 days in culture. Synapsin IIa also increased the amplitude of evoked synaptic currents by 2.3-fold in 2-day cultures. The evoked synaptic current amplitudes of syn IIa (+) synapses had a lower coefficient of variation indicating a more stable evoked response. These enhanced synaptic activities were independent of the presence or absence of the protein in the postsynaptic muscle cell. The findings indicate a role for synapsin IIa in synapse maturation.

We have isolated and characterized two new protein kinase C (PKC) genes from D. melanogaster. One, dPKC98F, maps to chromosome region 98F and displays over 60% amino acid sequence identity with members of a recently described "PKC-related" subfamily in mammals. The other, dPKC53E(ey), maps to region 53E 4–7 on the second chromosome and lies within 50 kb of a PKC gene previously characterized (dPKC). While dPKC98F transcripts are expressed throughout development, expression of the two genes mapping at cytogenetic location 53E is primarily in adults. dPKC98F and the previously reported 53E gene are transcribed predominantly in brain tissue. In contrast, dPKC53E(ey) is transcribed only in photoreceptor cells. We will discuss the significance of this tissue-specific localization with regard to phototransduction.

Proteins that are related evolutionarily may have diverged at the level of primary amino acid sequence while maintaining similar secondary structures. Computer analysis has been used to compare the open reading frames of the hepatitis B virus to those of the woodchuck hepatitis virus at the level of amino acid sequence, and to predict the relative hydrophilic character and the secondary structure of putative polypeptides. Similarity is seen at the levels of relative hydrophilicity and secondary structure, in the absence of sequence homology. These data reinforce the proposal that these open reading frames encode viral proteins. Computer analysis of this type can be more generally used to establish structural similarities between proteins that do not share obvious sequence homology as well as to assess whether an open reading frame is fortuitous or codes for a protein.