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Neuropharmacology Division Postdoctoral Scientist Award Finalists
Tuesday April 21, 2009 - New Orleans Convention Center, Room 206
Chair: Christian C. Felder
Schedule of Presentations
3:00 PM – 3:25 PM
Keynote Address
Perspectives on the postdoctoral experience in the pharmaceutical industry Christian C. Felder, Eli Lil y and Co.
Each presenter will give a 15-minute oral presentation, followed by a 10-minute discussion.
3:25 PM – 3:50 PM
Caveats of proteomics approaches in identifying novel spinophilin interacting proteins (Abstract 581.9) A.J. Baucum II, A-J.L. Ham, R.J. Colbran, Vanderbilt University
3:50 PM – 4:15 PM
Reduced microscopic GABAR sensitivity in rat hippocampal CA1 neurons during benzodiazepine withdrawal is reversed by CaMKII inhibition (Abstract 760.13) P. Das, L.J. Greenfield, Jr., E.I. Tietz, University of Toledo Col ege of Medicine
4:15 PM – 4:40 PM
The cannabinoid CB1 receptor system modulates behavioral phenomena predictive of relapse during extinction of self-administration in mice (Abstract 58837) S.J. Ward, M. Rosenberg, L. Dykstra, E. Walker, Temple University School of
Medicine and University of North Carolina at Chapel Hil

4:40 PM – 5:05 PM
Ex vivo and in vivo changes in function of the serotonin 2C receptor due to RNA editing (Abstract 842.2) C.E. Canal, e. E. Watt, E. Sanders-Bush, Vanderbilt University
5:05 PM – 5:30 PM
Beyond Prozac: generation and characterization of SSRI insensitive transgenic mice (Abstract 942.7) B.J. Thompson, T. Jessen, L.K. Henry, K.L. Gamble, P.J. Chisnell, D.G.
McMahon, R.D. Blakel,
Vanderbilt University and University of North Dakota
ABSTRACTS IN ORDER OF PRESENTATIONS
Caveats of proteomics approaches in identifying novel spinophilin interacting proteins (Abstract
581.9)

A.J. Baucum II, A-J.L. Ham, R.J. Colbran, Vanderbilt University
Spinophilin is an F-actin and protein phosphatase 1 (PP1) binding protein that targets PP1 to the postsynaptic density and to various dendritic substrates. PP1 binding to spinophilin is enhanced in an animal model of parkinsonism. Several other known spinophilin associated proteins (SpAPs) were not detected in spinophilin complexes isolated from rat or mouse striatum. Therefore, a de novo shotgun proteomics strategy was developed to identify SpAps. Complexes specifical y isolated using goat, mouse or rabbit spinophilin antibodies contained spinophilin, PP1, and neurabin (a known SpAP), as wel as novel candidate SpAPs. We selected one of the novel proteins for further validation: the thousand and one amino acid kinase (TAO1). While TAO1 and spinophilin were detected in the reciprocal precipitations, and the amount of co-immunoprecipitation was reduced fol owing dopamine depletion, TAO1 was also present in spinophilin immunoprecipitates isolated from extracts of spinophilin knockout (KO) mice. Therefore, to identify spinophilin-selective interactions, spinophilin was immunoprecipitated from both WT and KO animals, using multiple antibodies, and these samples were analyzed by shotgun proteomics. Multiple proteins were present in complexes isolated from WT, but not KO, tissue and studies are ongoing to validate these interactions.
Reduced microscopic GABAR sensitivity in rat hippocampal CA1 neurons during benzodiazepine
withdrawal is reversed by CaMKII inhibition (Abstract 760.13)

P. Das, L.J. Greenfield, Jr., E.I. Tietz, University of Toledo Col ege of Medicine
Two days after 1-week oral treatment with the benzodiazepine (BZ), flurazepam (FZP), L-type voltage-gated channel (L-VGCC) whole-cel Ca2+ current density is doubled and γ-amino butyric acid type A receptor (GABAR) single-channel open probability (Po) is reduced in cel -attached patches from dissociated hippocampal CA1 neurons. Blockade of Ca2+ influx by systemic injection of the L-VGCC antagonist, nimodipine 1 day after FZP treatment, restored sensitivity of FZP neurons to 5 μM GABA, increasing Po from 0.04 to 0.54 after 2 days to near control levels (0.68). We hypothesized that BZ-induced elevation of [Ca2+]i may suppress GABAR function by activating Ca2+/calmodulin-dependent protein kinase II (CaMKII). Single-channel recordings were obtained after slice pre-incubation with membrane-permeant CaMKII inhibitors. At 1 μM GABA, Po in control patches was 0.19 while no openings were observed in FZP patches. After pre-incubation with KN93, Po in FZP patches was 0.73. No openings were seen with the inactive analog KN92. Pre-incubation with mAIP, activated ‘silent’ single channels in FZP patches (Po=0.69) and increased the basal response of control patches (Po=0.57). Membrane-impermeant AIP and the PKC inhibitor, chelerythrine had no effect. Persistent activation of CaMKII is required to maintain basal GABAR function and for the BZ-induced, negative modulation of GABAR function associated with BZ tolerance. NIDA grants R01-DA018342 and RO1-DA04075 (EIT) The cannabinoid CB1 receptor system modulates behavioral phenomena predictive of relapse
during extinction of self-administration in mice (Abstract 58837)

S.J. Ward, M. Rosenberg, L. Dykstra, E. Walker, Temple University School of Medicine and University of
North Carolina at Chapel Hil

The endogenous cannabinoid system plays a defined role in the extinction of fear conditioning and spatial memory. It is also wel -established that manipulation of CB1 receptors modulates positively-reinforced behaviors such as drug self-administration in laboratory animals, but whether and how the CB1 system regulates extinction of such behaviors is poorly understood. We investigated whether the CB1 receptor antagonist SR141716 affects several extinction behaviors and/or the rate of extinction learning in mice trained to self-administer palatable food (32% corn oil or 50% Ensure p.o.) or cocaine (0.3 mg/kg/inf i.v.). Fol owing stable self-administration behavior, mice were pretreated prior to daily extinction sessions with 3.0 mg/kg SR141716 or vehicle, and effects on initial extinction burst responding, spontaneous recovery, condi toned stimulus (CS) - or prime-induced reinstatement, and daily extinction rates were assessed. SR141716 pretreatment attenuated initial burst responding, spontaneous recovery, and CS- and prime- induced reinstatement, and SR141716-treated mice also required fewer trials to reach daily extinction criterion than vehicle-treated mice. Taken together, these data suggest that in addition to attenuating the primary reinforcing effects of both palatable foods and drugs of abuse, CB1 receptor antagonism can attenuate relapse behaviors during extinction and enhance extinction learning.
Ex vivo and in vivo changes in function of the serotonin 2C receptor due to RNA editing (Abstract
842.2)

C.E. Canal, e. E. Watt, E. Sanders-Bush, Vanderbilt University
In vitro, RNA editing of the serotonin 2C receptor (5HT2CR) alters its signal transduction efficiency. Ful y edited (VGV) 5HT2CR's show low constitutive and agonist-stimulated activity, whereas non-edited (INI) 5HT2CR's exhibit the converse pattern. To test whether 5HT2CR editing alters its function ex vivo, 125-I DOI and 3-H mesulergine autoradiography was performed on brain sections from mice solely expressing either the 5HT2C-INI or 5HT2C-VGV receptor. Similar to in vitro observations, VGV receptors exhibited decreased, whereas INI receptors exhibited increased high affinity G-protein coupling relative to wild-type (WT) receptors. In addition, relative to WT mice, INI expressing mice showed greater increases in plasma corticosterone fol owing treatment with the 5HT2CR agonist, MK-212. INI expressing mice did not show alterations in expression of glucocorticoid receptor, suggesting that changes in the stress circuit did not account for the differences in 5HT2CR agonist-induced corticosterone release. These data show that editing of the 5HT2CR alters its function in vivo. Supported by: NIH T32-MH065215 (CEC) and R01-MH34007 (ESB) Beyond Prozac: generation and characterization of SSRI insensitive transgenic mice (Abstract
942.7)

B.J. Thompson, T. Jessen, L.K. Henry, K.L. Gamble, P.J. Chisnell, D.G. McMahon, R.D. Blakel,
Vanderbilt University and University of North Dakota
Altered activity of the serotonin transporter (SERT) transporter has been hypothesized to contribute to major depression, anxiety, obsessive compulsive disorder, and autism. A principal component of this theory is the belief that SERTs are the primary target for selective serotonin reuptake inhibitor (SSRI)-class antidepressants, as wel as many tricyclic antidepressants (TCA’s). Cross-species analysis of SERT has identified a substitution (I172M) that, in vitro, results in a dramatic loss of affinity for cocaine, several SSRI’s and TCA’s with no appreciable effect on 5HT transport (Henry, L.K., et al., 2006). We have introduced the I172M substitution into the SERT locus of 129S6 mice by homologous recombination. As predicted this substitution has no impact on 5HT transport and these animals exhibit no overt behavioral phenotype. Behavioral studies revealed a reduction in responsiveness to citalopram in the tail suspension test, as wel as reduced responsiveness to fluoxetine and citalopram in the forced swim test. Ex vivo analysis of forebrain synaptosomal 5HT transport from mice carrying this substitution demonstrate ~1,000 fold shift in citalopram potency, ~12 fold shift in fluoxetine potency and ~32 fold shift in cocaine potency. In contrast, paroxetine displayed less than a 2 fold shift in potency. These findings are complemented by ex vivo midbrain slice recordings of serotonergic neurons in the dorsal raphe, which show a significant shift in SSRIs induced autoinhibition of neuronal firing. Ongoing efforts aim to define the ful impact of the I172M mutation on in vivo and ex vivo SERT pharmacology as wel as the effect this loss of SSRI potency has on SSRI induced behavior.

Source: http://www.aspet.org/uploadedFiles/Divisions_and_Chapters/ASPET_Divisions/Neuropharmacology/Content/Program-Postdoctoral%20Scientist%20Award%20Finalists.pdf

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