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1.  RiboTag is a flexible tool for measuring the translational state of targeted cells in heterogeneous cell cultures 
BioTechniques  2015;58(6):308-317.
Primary neuronal cultures are a useful tool for measuring pharmacological- and transgene-regulated gene expression; however, accurate measurements can be confounded by heterogeneous cell-types and inconsistent transfection efficiency. Here we describe our adaptation of a ribosomal capture strategy that was designed to be used in transgenic mice expressing tagged ribosomal subunits (RiboTag) in specific cell types, thereby allowing measurement of translating RNA from desired cell types within complex tissues. Using this strategy we were able to isolate and analyze neuron-specific RNA despite the presence of glia by co-transfecting experimental plasmids with plasmids that selectively express RiboTag in neurons. RiboTag immunoprecipitation was capable of recovering high integrity RNA from small numbers of transfected cells that can then be interrogated by a variety of methods (e.g. RT-qPCR, PCR array, RNAseq) and compared to basal RNA expression of the entire culture. Additionally, we demonstrate how co-transfection of RiboTag with sh-RNA constructs can validate and accurately assess the degree of gene expression knockdown, and how RiboTag can be used to measure receptor-mediated gene regulation with transiently expressed DREADD-receptors. RiboTag co-transfection represents a convenient and powerful tool to isolate RNA from a specific subset of cultured cells with a variety of applications for experiments in vitro.
PMCID: PMC4467021  PMID: 26054767
Cell-type specific gene expression; translational profiling; RiboTag; TRAP; primary neuronal cultures
2.  Effects of a Dopamine Agonist on the Pharmacodynamics of Levodopa in Parkinson Disease 
Archives of Neurology  2010;67(1):27-32.
Treatment of Parkinson disease commonly includes levodopa and dopamine agonists; however, the interaction of these 2 drugs is poorly understood.
To examine the effects of a dopamine agonist on the motor response to levodopa.
Double-blind, randomized, placebo-controlled, crossover clinical trial.
Ambulatory academic referral center.
Thirteen patients with idiopathic Parkinson disease taking levodopa and experiencing motor fluctuations and dyskinesia.
Eligible individuals were randomly assigned to receive pramipexole dihydrochloride or placebo for 4 weeks followed by a 2-hour intravenous levodopa infusion on consecutive days at 2 rates and with blinded assessments. They were then crossed over to the alternate oral therapy for 4 weeks followed by levodopa infusion and reassessment.
Main Outcome Measures
Change in finger-tapping speed, measured using the area under the curve (AUC) for finger taps per minute across time; peak finger-tapping speed; duration of response; time to “ON” (defined as a 10% increase in finger-tapping speed above baseline); walking speed; and dyskinesia AUC.
Pramipexole with levodopa infusion increased finger-tapping speed beyond the change in baseline by a mean (SE) of 170 (47.2) per minute×minutes (P=.006) and more than doubled the AUC for finger-tapping speed. Pramipexole increased peak finger-tapping speed by a mean (SE) of 18 (8.5) taps per minute (P=.02) and improved mean (SE) walking speed (15.9 [0.70] vs 18.9 [0.70] seconds, P=.004). Pramipexole prolonged duration of response after levodopa infusion and shortened time to ON. Pramipexole increased mean (SE) baseline dyskinesia scores (26.0 [5.85] vs 12.1 [5.85] points, P = .05) and peak dyskinesia scores with levodopa infusion.
Pramipexole augmented the motor response to levodopa beyond a simple additive effect and increased the severity of levodopa-induced dyskinesia. When considering a combination of these therapies, an appropriate balance should be maintained regarding gain of motor function vs worsening of dyskinesia.
Trial Registration Identifier: NCT00666653
PMCID: PMC3390306  PMID: 20065126
3.  Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement 
Neuron  2011;72(5):721-733.
Currently there is no general approach for achieving specific optogenetic control of genetically-defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically-restricted recombinase-driver rat lines suitable for driving gene expression in specific cell-types, expressing Cre recombinase under control of large genomic regulatory regions (200–300 Kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell-types. We additionally developed methods for utilizing optogenetic tools in freely-moving rats, and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.
PMCID: PMC3282061  PMID: 22153370
4.  Cholinergic Interneurons Control Local Circuit Activity and Cocaine Conditioning 
Science (New York, N.Y.)  2010;330(6011):1677-1681.
Cholinergic neurons are widespread, and pharmacological modulation of acetylcholine receptors affects numerous brain processes, but such modulation entails side effects due to limitations in specificity for receptor type and target cell. As a result, causal roles of cholinergic neurons in circuits have been unclear. We integrated optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology to probe the cholinergic interneurons of the nucleus accumbens by direct excitation or inhibition. Despite representing less than 1% of local neurons, these cholinergic cells have dominant control roles, exerting powerful modulation of circuit activity. Furthermore, these neurons could be activated by cocaine, and silencing this drug-induced activity during cocaine exposure (despite the fact that the manipulation of the cholinergic interneurons was not aversive by itself) blocked cocaine conditioning in freely moving mammals.
PMCID: PMC3142356  PMID: 21164015
5.  Diffusion of Botulinum Toxins 
Tremor and Other Hyperkinetic Movements  2012;2:tre-02-85-417-1.
It is generally agreed that diffusion of botulinum toxin occurs, but the extent of the spread and its clinical importance are disputed. Many factors have been suggested to play a role but which have the most clinical relevance is a subject of much discussion.
This review discusses the variables affecting diffusion, including protein composition and molecular size as well as injection factors (e.g., volume, dose, injection method). It also discusses data on diffusion from comparative studies in animal models and human clinical trials that illustrate differences between the available botulinum toxin products (onabotulinumtoxinA, abobotulinumtoxinA, incobotulinumtoxinA, and rimabotulinumtoxinB).
Neither molecular weight nor the presence of complexing proteins appears to affect diffusion; however, injection volume, concentration, and dose all play roles and are modifiable. Both animal and human studies show that botulinum toxin products are not interchangeable, and that some products are associated with greater diffusion and higher rates of diffusion-related adverse events than others.
Each of the botulinum toxins is a unique pharmacologic entity. A working knowledge of the different serotypes is essential to avoid unwanted diffusion-related adverse events. In addition, clinicians should be aware that the factors influencing diffusion may range from properties intrinsic to the drug to accurate muscle selection as well as dilution, volume, and dose injected.
PMCID: PMC3570036  PMID: 23440162
Botulinum toxin; diffusion; spread; injection technique

Results 1-5 (5)