This study sought to determine the roles of D1 and D2 receptors in mediating gambling reinforcement in pathological gamblers and controls (n=24/group), and the influence of reward novelty on these effects. Subjects received D2 antagonist, haloperidol (3mg), or D1-D2 antagonist, fluphenazine (3mg) in a placebo-controlled, counterbalanced, two-session design. Incentive motivation and hedonic impact were assessed before and after a 15-min slot machine game. Haloperidol tended to increase pre-game motivation but reduce the priming effect of the slot machine, while fluphenazine increased positive mood ratings but reduced motivation to gamble. Haloperidol effects were stronger when it was received on the first session, while fluphenazine had stronger effects after prior drug-free exposure. Results suggest D1 signaling is central to reward expectancy and motivation to gamble, and that moderate stimulation increases positive affect while reducing motivation to gamble. D1 blockade may also enhance reinforcement of a familiar task by interfering with reward expectancy.

TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

Anxiety is a highly prevalent comorbidity in coronary artery disease (CAD) and confers increased risk of subsequent cardiac events and mortality. However, biological mechanisms of this relationship are not well understood. Ceramides are sphingolipids involved in inflammatory signaling and cell viability in the periphery and nervous system, and are implicated in pathophysiological mechanisms associated with anxiety. This study aimed to investigate relationships between plasma ceramide concentrations and anxiety symptomology as assessed by the Spielberger State-Trait Anxiety Inventory trait subscale (STAI-T) in CAD patients with linear regressions. High performance liquid chromatography coupled electrospray ionization tandem mass spectrometry was used to assay sphingolipid species. Plasma C22:0 ceramide (β=-0.232, p=0.018) concentrations and 8 other species of sphingolipids (SM18:0, SM20:1, C18:0, C20:0, C18:1, DHC22:0, LacC22:0, LacC24:1) were negatively correlated with STAI-T score when controlling for gender, BMI, and CES-D. Findings suggest specific sphingolipids to be potential markers for anxiety severity in CAD.

This study examined the effects of 30 minutes of cycle exercise at 65% V̇O2max on the pharmacokinetics of the S.S.R.I. sertraline. Blood samples were taken over 48 hours from 14 healthy males (23.9±2.5 years, 80.3±12.6 kilograms) following oral ingestion of a single 100 mg dose of sertraline. Participants completed two sertraline trials separated by at least two weeks; one trial while resting and the other trial with exercise as described above. With exercise, the absorption rate constant and volume of sertraline in the central compartment decreased, while the elimination half-life increased. Maximum concentration, time of maximum concentration, and area under the curve were unchanged. Fitness level had little impact on the concentration of sertraline, as compartmental modeling was unchanged when relative V̇O2max was added as a covariate. However, controlling for participant body weight improved the model estimate. These results indicate that acute aerobic exercise has the potential to change the concentration of sertraline in vivo.

Propofol is a widely in use anesthetic drug. Propofol’s abuse liability has been supported by many case reports and a few animal studies. However, propofol’s reinforcing properties have not yet been investigated in-depth. In this study, multiple aspects of propofol’s abuse-related behaviour were investigated using the drug self-administration model in rats. METHODS: Rats were subjected to propofol self-administration under a fixed ratio 1 (FR1) schedule and different aspects of propofol self-administration behaviour including acquisition, maintenance of the behaviour under a higher ratio schedule, extinction and reinstatement were investigated. RESULTS: Rats acquired propofol self-administration under a FR1 schedule. The acquired behaviour was maintained under a FR2 schedule, showed a modest variation over a range of doses, and was extinguished upon substitution of vehicle for propofol, showing no reinstatement using a range of priming doses of propofol. CONCLUSION: Propofol has abuse potential showing modest reinforcing properties under our experimental conditions.

Deep brain stimulation (DBS) has emerged as a potential intervention for treatment-resistant tardive dyskinesia (TD). Despite promising case reports, no consensus exists regarding optimal stimulation parameters, neuroanatomical target for DBS in TD, or mechanisms underlying its anti-dyskinetic effects. We used vacuous chewing movements (VCMs) in rats treated chronically with haloperidol (HAL) as a TD model to address some of these issues. We show that acute DBS applied to the subthalamic nucleus (STN) or the entopeduncular nucleus (EPN) suppresses VCMs without affecting locomotor activity. Using immediate early gene mapping with zif268 as an index of neuronal activity, we found that STN-DBS induced decreases in activity throughout the basal ganglia, whereas EPN-DBS increased activity in projection regions. While chemical inactivation of the STN or EPN with the GABAA agonist muscimol also suppressed VCMs, muscimol infusion did not mimic the changes in neuronal activity induced by DBS, suggesting that DBS is not equivalent to functional inactivation. We next examined the contribution of serotonin (5-HT) and dopamine (DA) to the anti-dyskinetic effects of DBS. Decreasing 5-HT transmission pharmacologically or with serotonergic lesions decreased VCMs. Using microdialysis and zif268 mapping, we determined that STN- but not EPN-DBS decreased 5-HT release and activity of raphe neurons. However, when the decrease in 5-HT induced by STN-DBS was prevented by pre-treating rats with fluoxetine or fenfluramine, we found that decreasing 5-HT is not necessary for the anti-dyskinetic effects of DBS. STN-DBS transiently increased striatal DA release in intact rats only, whereas EPN-DBS had no effect on DA release. Moreover, pharmacologically elevating DA levels did not suppress VCMs. Together these findings lead us to conclude that increased DA release does not contribute to the anti-dyskinetic effects of DBS. Finally, we compared depressive- and anxiety-like behaviours induced by chronic DBS of the EPN and STN, since adverse psychiatric effects of DBS have become a significant clinical concern. STN-DBS but not EPN-DBS induced depressive-like behaviour in a learned helplessness task. We established that the chronic HAL VCM model preparation may be used to explore mechanisms underlying anti-dyskinetic and psychiatric effects of DBS, and provided the first investigations into these mechanisms.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.