Utilizing Miniature Fluorescence Microscopy to Image Hippocampal Place Cell Ensemble Function in Thy1.GCaMP6f Transgenic Mice.
Authors: Indersmitten T, Berdyyeva T, Aluisio L, Lovenberg T, Bonaventure P, Wyatt RM
Abstract
Imaging neuronal activity in awake behaving mice with miniature fluorescence microscopes requires the implementation of a variety of procedures. Surgeries are performed to gain access to the cell population of interest and to implant microscope components. After a recovery period, mice are trained to exhibit a desired behavior. Finally, neuronal activity is imaged and synchronized with that behavior. To take full advantage of the technology, selection of the calcium indicator and experimental design must be caref...
Source: Current Protocols in Pharmacology - August 22, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Enzyme Assays for JmjC-Domain-Containing Lysine Histone Demethylases (JmjC-KDMs).
Authors: Tarhonskaya H, Tumber A, Kawamura A, Schofield CJ
Abstract
Histone modifications, including lysine methylation marks on histone tails, modulate the accessibility of genes for transcription. Changes in histone tail methylation patterns can cause transcriptional activation or repression. The dynamic regulation of lysine methylation patterns is enabled by two distinct groups of enzymes: histone methyltransferases (KMTs) and demethylases (KDMs). The Jumonji C (JmjC) domain-containing lysine histone demethylases (JmjC-KDMs) alter the methylation levels of histone tails by removing tri-, di-, or mono-me...
Source: Current Protocols in Pharmacology - July 26, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease.
Authors: Zolotarjova NI, Wynn R
Abstract
Bromodomains are protein domains that recognize acetylated lysine residues and are important for recruiting a large number of protein and multiprotein complexes to sites of lysine acetylation. They play an important role in chromatin biology and are popular targets for drug discovery. Compound screening in this area requires the use of biochemical assays to assess the binding potency of potential drug candidates. Foremost among the efforts to target bromodomains are those aimed at identifying compounds that interact with the bromodomain and extra-terminal domain (BE...
Source: Current Protocols in Pharmacology - July 26, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Three Decades of Chloride Intracellular Channel Proteins: From Organelle to Organ Physiology.
Authors: Gururaja Rao S, Ponnalagu D, Patel NJ, Singh H
Abstract
Intracellular organelles are membranous structures central for maintaining cellular physiology and the overall health of the cell. To maintain cellular function, intracellular organelles are required to tightly regulate their ionic homeostasis. Any imbalance in ionic concentrations can disrupt energy production (mitochondria), protein degradation (lysosomes), DNA replication (nucleus), or cellular signaling (endoplasmic reticulum). Ionic homeostasis is also important for volume regulation of intracellular organelles and is maintained by catio...
Source: Current Protocols in Pharmacology - July 26, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Quantitative Intracerebral Microdialysis Studies to Determine Unbound Extracellular Fluid Drug Concentrations in Discrete Areas of the Brain.
Authors: Durk MR
Abstract
Intracerebral microdialysis is a powerful technique for quantifying unbound brain extracellular fluid concentrations of drugs and drug candidates over time in live, conscious, freely moving animals. The results provide crucial information on the brain penetrance of the administered agent. This unit details the surgical procedures, study planning and execution, and data analysis required for a brain microdialysis study following intravenous infusion of a test substance to steady state. The accumulated data makes possible the determination of the area under the concentration-time cu...
Source: Current Protocols in Pharmacology - July 26, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Measuring Histone Deacetylase Inhibition in the Brain.
Authors: Reddy DS, Wu X, Golub VM, Dashwood WM, Dashwood RH
Abstract
Histone deacetylases (HDACs) represent a family of enzymes that are targets for epigenetic modulation of genomic activity and may be beneficial in the treatment of many diseases, including cancer and central nervous system disorders. In animal models, HDAC inhibitors have neuroprotective, antiepileptogenic, and antidepressant effects. Assaying HDAC activity provides a robust method for identifying HDAC inhibitors and for assessing their effects under various physiological conditions or after pathological insults. In this unit, a simple an...
Source: Current Protocols in Pharmacology - June 27, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers.
Authors: Klaerke DA, Tejada MLA, Christensen VG, Lassen M, Pedersen PA, Calloe K
Abstract
Detergent-solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar "black lipid membranes" formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step-by-step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for pla...
Source: Current Protocols in Pharmacology - June 27, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of Genetically Engineered Mouse Models of Prostate Cancer and Their Applications in Drug Discovery.
Authors: Civenni G, Carbone GM, Catapano CV
Abstract
Prostate cancer (PCa) is the most common malignant visceral neoplasm in males in Western countries. Despite progress made in the early treatment of localized malignancies, there remains a need for therapies effective against advanced forms of the disease. Genetically engineered mouse (GEM) models are valuable tools for addressing this issue, particularly in defining the cellular and molecular mechanisms responsible for tumor initiation and progression. While cell and tissue culture systems are important models for this purpose as well, they cannot recapi...
Source: Current Protocols in Pharmacology - June 27, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Reagent Validation to Facilitate Experimental Reproducibility.
Authors: Williams M
Abstract
Many results reported in the biomedical research literature cannot be independently reproduced, undermining the basic foundations of science. This overview is intended for researchers who are committed to improving the quality and integrity of biomedical science by raising awareness of both the sources of irreproducibility, and activities specifically targeted to address the issue. The irreproducibility of biomedical research is due to a variety of factors, known and unknown, that markedly influence experimental outcomes. Among the known factors are inadequate training or mento...
Source: Current Protocols in Pharmacology - June 27, 2018 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Validation of miRNA-Mediated Gene Expression Linked to Drug Metabolism.
Authors: Shenoda BB, Ramanathan S, Ajit SK
Abstract
Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarker...
Source: Current Protocols in Pharmacology - December 21, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT).
Authors: Aggarwal S, Mortensen OV
Abstract
Detailed in this unit are protocols for studying the in vitro uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (KM ) and maximal velocity (Vmax ) of radiolabeled DA uptake into cells; concentration-response assays to measure the potencies (IC50 /Ki values) of test compounds as transport inhibitors; and an ef...
Source: Current Protocols in Pharmacology - December 21, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of microRNA Modulation in Analgesic Research.
Authors: Ramanathan S, Shenoda BB, Ajit SK
Abstract
MicroRNA(miRNA)-mediated gene regulation underlies cellular processes, playing an important role in homeostasis and diseases. The expression and function of miRNAs are altered by various pharmacological agents, with differences in the endogenous levels of miRNAs influencing drug efficacy and toxicity. Thus, miRNA levels could be a biomarker for predicting treatment response, efficacy, and safety. In addition, elucidating the mechanistic significance of miRNA alterations can aid in the identification of therapeutic targets and patient selection, and guide ...
Source: Current Protocols in Pharmacology - December 21, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of Monoamine Transporters.
Authors: Aggarwal S, Mortensen OV
Abstract
The dopamine (DAT), serotonin (SERT), and norepinephrine (NET) transporters, which are collectively referred to as monoamine transporters (MATs), play significant roles in regulating the neuronal response to these neurotransmitters. MATs terminate the action of these neurotransmitters by translocating them from the synaptic space into the presynaptic neurons. These three transmitters are responsible for controlling a number of physiological, emotional, and behavioral functions, with their transporters being the site of action of drugs employed for the treatment of...
Source: Current Protocols in Pharmacology - December 21, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Histone Acetylation Assay.
Authors: Brown JAL
Abstract
Acetylation is a core cellular process involved in maintaining genomic integrity, gene regulation, and metabolism. Histone acetyltransferases (HATs) are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. Perturbations of cellular acetylation profiles have been associated with a variety of disease states, including cancer. Changes in acetylation profiles can be achieved by mechanisms associated with acetyltransferases, such as gene down-regulation or alterations in the activity of key acetyltransferase enzymes. An ...
Source: Current Protocols in Pharmacology - December 21, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Rodent Test of Attention and Impulsivity: The 5-Choice Serial Reaction Time Task.
Authors: Higgins GA, Silenieks LB
Abstract
The 5-choice serial reaction time task (5-CSRTT) is employed extensively to measure attention in rodents. The assay involves animals trained to respond to a brief, unpredictable visual stimulus presented in one of five locations. The effects of experimental manipulations on response speed and choice accuracy are measured, and each related to attentional performance. The 5-CSRTT is also used to measure motor impulsivity. Adapted from a human task, the 5-CSRTT can be employed with rodents or primates, highlighting its translational value. Another strength of this pr...
Source: Current Protocols in Pharmacology - September 12, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
