Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens.

Despite abundant expression of DNA methyltransferases (Dnmts) in brain, the regulation and behavioral role of DNA methylation remain poorly understood. We found that Dnmt3a expression was regulated in mouse nucleus accumbens (NAc) by chronic cocaine use and chronic social defeat stress. Moreover, NAc-specific manipulations that block DNA methylation potentiated cocaine reward and exerted antidepressant-like effects, whereas NAc-specific Dnmt3a overexpression attenuated cocaine reward and was pro-depressant. On a cellular level, we found that chronic cocaine use selectively increased thin dendritic spines on NAc neurons and that DNA methylation was both necessary and sufficient to mediate these effects. These data establish the importance of Dnmt3a in the NAc in regulating cellular and behavioral plasticity to emotional stimuli

Endogenous cholinergic inputs and local circuit mechanisms govern the phasic mesolimbic dopamine response to nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.Peer reviewe

A validated stability-indicating HPLC method for determination of varenicline in its bulk and tablets

A simple, sensitive and accurate stability-indicating HPLC method has been developed and validated for determination of varenicline (VRC) in its bulk form and pharmaceutical tablets. Chromatographic separation was achieved on a Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm i.d., particle size 5 μm, maintained at ambient temperature) by a mobile phase consisted of acetonitrile and 50 mM potassium dihydrogen phosphate buffer (10:90, v/v) with apparent pH of 3.5 ± 0.1 and a flow rate of 1.0 ml/min. The detection wavelength was set at 235 nm. VRC was subjected to different accelerated stress conditions. The degradation products, when any, were well resolved from the pure drug with significantly different retention time values. The method was linear (r = 0.9998) at a concentration range of 2 - 14 μg/ml. The limit of detection and limit of quantitation were 0.38 and 1.11 μg/ml, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 2%. The accuracy of the method was proved; the mean recovery of VRC was 100.10 ± 1.08%. The proposed method has high throughput as the analysis involved short run-time (~ 6 min). The method met the ICH/FDA regulatory requirements. The proposed method was successfully applied for the determination of VRC in bulk and tablets with acceptable accuracy and precisions; the label claim percentages were 99.65 ± 0.32%. The results demonstrated that the method would have a great value when applied in quality control and stability studies for VRC

Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

Mechanisms Involved in Nicotinic Acetylcholine Receptor-Induced Neurotransmitter Release from Sympathetic Nerve Terminals in the Mouse Vas Deferens

Prejunctional nicotinic acetylcholine receptors (nAChRs) amplify postganglionic sympathetic neurotransmission, and there are indications that intraterminal Ca2+ stores might be involved. However, the mechanisms by which nAChR activation stimulates neurotransmitter release at such junctions is unknown. Rapid local delivery (picospritzing) of the nAChR agonist epibatidine was combined with intracellular sharp microelectrode recording to monitor spontaneous and field-stimulation-evoked neurotransmitter release from sympathetic nerve terminals in the mouse isolated vas deferens. Locally applied epibatidine (1 µM) produced ‘epibatidine-induced depolarisations’ (EIDs) that were similar in shape to spontaneous excitatory junction potentials (SEJPs) and were abolished by nonselective nAChR antagonists and the purinergic desensitizing agonist α,β-methylene ATP. The amplitude distribution of EIDs was only slightly shifted towards lower amplitudes by the selective α7 nAChR antagonists α-bungarotoxin and methyllcaconitine, the voltage-gated Na+ channel blocker tetrodotoxin or by blocking voltage-gated Ca2+ channels with Cd2+. Lowering the extracellular Ca2+ concentration reduced the frequency of EIDs by 69%, but more surprisingly, the Ca2+-induced Ca2+ release blocker ryanodine greatly decreased the amplitude (by 41%) and the frequency of EIDs by 36%. Ryanodine had no effect on electrically-evoked neurotransmitter release, paired-pulse facilitation, SEJP frequency, SEJP amplitude or SEJP amplitude distribution. These results show that activation of non-α7 nAChRs on sympathetic postganglionic nerve terminals induces high-amplitude junctional potentials that are argued to represent multipacketed neurotransmitter release synchronized by intraterminal Ca2+-induced Ca2+ release, triggered by Ca2+ influx directly through the nAChR. This nAChR-induced neurotransmitter release can be targeted pharmacologically without affecting spontaneous or electrically-evoked neurotransmitter release

Behavioral Sequence Analysis Reveals a Novel Role for ß2* Nicotinic Receptors in Exploration

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and modulate neuronal function in most mammalian brain structures. The contribution of defined nAChR subunits to a specific behavior is thus difficult to assess. Mice deleted for ß2-containing nAChRs (ß2−/−) have been shown to be hyperactive in an open-field paradigm, without determining the origin of this hyperactivity. We here develop a quantitative description of mouse behavior in the open field based upon first order Markov and variable length Markov chain analysis focusing on the time-organized sequence that behaviors are composed of. This description reveals that this hyperactivity is the consequence of the absence of specific inactive states or “stops”. These stops are associated with a scanning of the environment in wild-type mice (WT), and they affect the way that animals organize their sequence of behaviors when compared with stops without scanning. They characterize a specific “decision moment” that is reduced in ß2−/− mutant mice, suggesting an important role of ß2-nAChRs in the strategy used by animals to explore an environment and collect information in order to organize their behavior. This integrated analysis of the displacement of an animal in a simple environment offers new insights, specifically into the contribution of nAChRs to higher brain functions and more generally into the principles that organize sequences of behaviors in animals

Alternative Ii-independent antigen-processing pathway in leukemic blasts involves TAP-dependent peptide loading of HLA class II complexes

During HLA class II synthesis in antigen-presenting cells, the invariant chain (Ii) not only stabilizes HLA class II complexes in the endoplasmic reticulum, but also mediates their transport to specialized lysosomal antigen-loading compartments termed MIICs. This study explores an alternative HLA class II presentation pathway in leukemic blasts that involves proteasome and transporter associated with antigen processing (TAP)-dependent peptide loading. Although HLA-DR did associate with Ii, Ii silencing in the human class II-associated invariant chain peptide (CLIP)-negative KG-1 myeloid leukemic cell line did not affect total and plasma membrane expression levels of HLA-DR, as determined by western blotting and flow cytometry. Since HLA-DR expression does require peptide binding, we examined the role of endogenous antigen-processing machinery in HLA-DR presentation by CLIP− leukemic blasts. The suppression of proteasome and TAP function using various inhibitors resulted in decreased HLA-DR levels in both CLIP− KG-1 and ME-1 blasts. Simultaneous inhibition of TAP and Ii completely down-modulated the expression of HLA-DR, demonstrating that together these molecules form the key mediators of HLA class II antigen presentation in leukemic blasts. By the use of a proteasome- and TAP-dependent pathway for HLA class II antigen presentation, CLIP− leukemic blasts might be able to present a broad range of endogenous leukemia-associated peptides via HLA class II to activate leukemia-specific CD4+ T cells