Group II mGluRs suppress hyperexcitability in mouse and human nociceptors.

We introduce a strategy for preclinical research wherein promising targets for analgesia are tested in rodent and subsequently validated in human sensory neurons. We evaluate group II metabotropic glutamate receptors, the activation of which is efficacious in rodent models of pain. Immunohistochemical analysis showed positive immunoreactivity for mGlu2 in rodent dorsal root ganglia (DRG), peripheral fibers in skin, and central labeling in the spinal dorsal horn. We also found mGlu2-positive immunoreactivity in human neonatal and adult DRG. RNA-seq analysis of mouse and human DRG revealed a comparative expression profile between species for group II mGluRs and for opioid receptors. In rodent sensory neurons under basal conditions, activation of group II mGluRs with a selective group II agonist produced no changes to membrane excitability. However, membrane hyperexcitability in sensory neurons exposed to the inflammatory mediator prostaglandin E2 (PGE2) was prevented by (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC). In human sensory neurons from donors without a history of chronic pain, we show that PGE2 produced hyperexcitability that was similarly blocked by group II mGluR activation. These results reveal a mechanism for peripheral analgesia likely shared by mice and humans and demonstrate a translational research strategy to improve preclinical validation of novel analgesics using cultured human sensory neurons.

Epigenetic response to environmental stress: Assembly of BRG1-G9a/GLP-DNMT3 repressive chromatin complex on Myh6 promoter in pathologically stressed hearts.

Chromatin structure is determined by nucleosome positioning, histone modifications, and DNA methylation. How chromatin modifications are coordinately altered under pathological conditions remains elusive. Here we describe a stress-activated mechanism of concerted chromatin modification in the heart. In mice, pathological stress activates cardiomyocytes to express Brg1 (nucleosome-remodeling factor), G9a/Glp (histone methyltransferase), and Dnmt3 (DNA methyltransferase). Once activated, Brg1 recruits G9a and then Dnmt3 to sequentially assemble repressive chromatin-marked by H3K9 and CpG methylation-on a key molecular motor gene (Myh6), thereby silencing Myh6 and impairing cardiac contraction. Disruption of Brg1, G9a or Dnmt3 erases repressive chromatin marks and de-represses Myh6, reducing stress-induced cardiac dysfunction. In human hypertrophic hearts, BRG1-G9a/GLP-DNMT3 complex is also activated; its level correlates with H3K9/CpG methylation, Myh6 repression, and cardiomyopathy. Our studies demonstrate a new mechanism of chromatin assembly in stressed hearts and novel therapeutic targets for restoring Myh6 and ventricular function. The stress-induced Brg1-G9a-Dnmt3 interactions and sequence of repressive chromatin assembly on Myh6 illustrates a molecular mechanism by which the heart epigenetically responds to environmental signals. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Astrocytes contribute to gamma oscillations and recognition memory.

Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.

Human sensory neurons: Membrane properties and sensitization by inflammatory mediators.

Biological differences in sensory processing between human and model organisms may present significant obstacles to translational approaches in treating chronic pain. To better understand the physiology of human sensory neurons, we performed whole-cell patch-clamp recordings from 141 human dorsal root ganglion (hDRG) neurons from 5 young adult donors without chronic pain. Nearly all small-diameter hDRG neurons (<50 µm) displayed an inflection on the descending slope of the action potential, a defining feature of rodent nociceptive neurons. A high proportion of hDRG neurons were responsive to the algogens allyl isothiocyanate (AITC) and ATP, as well as the pruritogens histamine and chloroquine. We show that a subset of hDRG neurons responded to the inflammatory compounds bradykinin and prostaglandin E2 with action potential discharge and show evidence of sensitization including lower rheobase. Compared to electrically evoked action potentials, chemically induced action potentials were triggered from less depolarized thresholds and showed distinct afterhyperpolarization kinetics. These data indicate that most small/medium hDRG neurons can be classified as nociceptors, that they respond directly to compounds that produce pain and itch, and that they can be activated and sensitized by inflammatory mediators. The use of hDRG neurons as preclinical vehicles for target validation is discussed.

Whole blood leukocytes isolation with microfabricated filter for cell analysis.

The flow cytometric analysis of leukocytes in whole blood usually requires isolation of leukocytes from other components of whole blood. Density gradient centrifugation and red blood cell lysis are the most commonly used methods to separate leukocytes but come with significant limitations. We report the results of the evaluation of a microfabricated filtration device for blood preparation that separates erythrocytes from leukocytes based on their size and mechanical properties. The microfabricated filter evaluated here requires a rapid and simple procedure and results in high leukocytes recovery without introducing bias among the leukocyte subpopulations. The filter removes erythrocytes, platelets, plasma proteins, and unbound staining reagent. This gentle filtration process produces very clean stained leukocytes for cytometric analysis without any apparent damage to leukocytes.

Automated voltage-clamp technique.

The voltage-clamp electrophysiology method is the gold standard for measuring the function of ion channels. In the past, this technique has had limited applicability in pharmaceutical drug discovery because of its low throughput, steep learning curve, and challenges in standardization of the experiments. Recently, new electrophysiology platforms have been developed, which are based on the use of planar electrodes. One key advantage of the new electrode geometry is that it makes the process of cell-to-electrode sealing amenable to automation, thus increasing the throughput and significantly reducing the skill-set needed to run the experiments. The further addition of computer-controlled fluidics, voltage-clamping electronics, and automated data handling makes it possible to perform multiple electrophysiology experiments in parallel with a high degree of consistency and in completely automated mode. Among the new offerings for automated voltage clamp, one of the systems, PatchXpress/Sealchip, is quickly becoming the new gold standard for the quantification of ion channel function. In this chapter, we provide an overview of the new planar patch-clamping platforms and describe how electrophysiology experiments are performed on the PatchXpress/Sealchip automated system.

Efficient characterization of use-dependent ion channel blockers by real-time monitoring of channel state.

Ion channels are important therapeutic targets for the treatment of a variety of conditions. Among ion channel blocking agents, use-dependent inhibitors can be especially effective therapeutic agents. Use dependence allows the selective inhibition of hyperactive neurons or tachycardiac myocytes, while minimizing effects on cells with normal activity. For voltage-gated channels, the use-dependent compounds typically bind to and inhibit a particular kinetic state that is induced by specific voltage changes. Drug discovery programs that focus on this class of drugs need to rank the use dependence of the compounds. A meaningful comparison among different molecules requires voltage clamp-based assays with continuous voltage control and compensation for or elimination of electrode drift-related effects. A method was developed based on automated electrophysiology in which voltage and frequency dependence of voltage-gated ion channel blockers can be compared using a protocol in which voltage error is compensated for in real time.

Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds.

Unintended inhibition of the cardiac potassium channel human ether-a-go-go-related gene (hERG) is considered the main culprit in drug-induced arrhythmias known as torsades de pointes. Electrophysiology is the most reliable in vitro screening method for identifying potential cardiac hERG liabilities, but only the recent advent of planar electrode-based voltage clamp electrophysiology promises sufficient throughput to support the drug testing needs of most drug discovery programs. We have assessed the reliability of this new format of the voltage clamp technology in measuring the activity of small molecules on the hERG channel. Based on the results herein of a screening against a panel of well-characterized hERG-active and -inactive molecules, we demonstrate that planar electrode electrophysiology, utilizing the Sealchip and PatchXpress technology platform (AVIVA Biosciences Corp., San Diego, CA), is comparable to traditional electrophysiology based on glass micropipettes in its reliability and data content. The new technology will allow significantly higher throughput and more thorough testing of pharmaceutical compounds.