Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

Manual patch clamp recordings offer direct insight into ion channel properties through the characterization of ion channel activity. Due to the high quality data it is considered the gold standard for ion channel research. Still, the limited throughput means that maximizing data from each cell, especially sensitive primary cells, is important.We have developed a patch clamp‐based assay for characterization fast‐acting ion channels, in this case exemplified by P2X, in primary dorsal root ganglion (DRG) neurons. This assay is based on the use of primary DRG neurons in culture as a cell model for chronic pain. These neurons retain their sensory functionality and remain responsive to thermal, mechanical and functional stimuli, and when supplemented with nerve growth factor (NGF) they can be used to mimic peripheral sensitization. The assay utilizes a microfluidic perfusion system, Dynaflow Resolve, to facilitate a stable recording situation and fast and programmable solution exchange. At the start of the experiment, compounds and buffer are loaded in the 16 wells of the Dynaflow Resolve chip. Micro‐channels connect each well to a recording chamber where the cells are added. To be able to provide fast solution exchange the DRG neuron is lifted using the patch pipette and positioned in front of the micro‐channel outlets. Then, the cell is scanned through the discrete flow zones formed outside the channels.A comparison between conventional bath perfusion and the automated Dynaflow Resolve system showed that both systems have similar capacity when it comes to obtaining successful high resistance seals. However, for collecting full 5‐8 point dose‐responses from single cells, the success rate for the Dynaflow Resolve superseded the conventional system. This enables a dramatically increased productivity when performing more complex pharmacological characterizations.

To overcome limitations for current automated patch-clamp systems, we have used a microfluidic approach to achieve rapid solution exchange around patch-clamped cells. A fully automated system has been developed, which provides all necessary plate, liquid and cell handling throughout an experiment. We have achieved 10-90% response times to glycine on glycine receptor expressing cells below 30ms. Results on several celland ion channel receptor types show good correspondence with reference measurements and literature for pharmaceutical parameters such as EC50.

Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.

Cellaxess®HT is a fully automated transfection system for reagent-free small interfering RNA (siRNA) delivery to a wide variety of primary and hard-to-transfect cell types with excellent transfection efficiencies and cell viabilities. CellaxessHT transfects cells directly in high-content analysis (HCA)-compatible 384-well microplates at a throughput of up to 50,000 wells per day. Because the system enables genomic screening of biologically relevant cell types, CellaxessHT can have an important role in identifying high-quality drug targets.

Ion channels are transm embrane proteins, found in virtually all cell types throughout the human body. Ion channels underlie neural communication, memory, behavior, every movement and heartbeat, and are as such prone to cause disease if malfunctioning. Therefore ion channels are very important targets in drug discovery. The gold standard technique for obtaining information on ion channel function with high information content and temporal resolution is patch-clamp. The technique measures the minute currents originating from the movement of ions across the cellular membrane, and enables determination of the potency and efficacy of a drug. However, patch-clamp suffers from serious throughput restrictions due to its laborious nature. To address the throughput problems we have developed a microfluidic chip containing 48 microchannels for an extremely rapid, sequential delivery of a large number of completely controlled solution environments to a lifted, patch-clamped cell. In this way, throughput is increased drastically compared to classical patch-clamp perfusion set-ups, with uncompromised data quality. The 48-microchannel chip has been used for the characterization of drugs affecting ligand-gated ion channels including agonists, antagonists and positive modulators with positive effects on both throughput and data quality.

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.

There has been enormous development in the field of microfluidic systems over the past decade(1-3). It has grown from a specialized area of research into a field engaging hundreds of research groups and a growing number of companies. The development of microfluidic systems was made possible by fabrication technologies developed in the microelectronics industry. This began in the late 1970s, when Terry et al.(4,5) demonstrated a gas chromatography system fabricated on a Si wafer, but received little attention until 1990, when Manz et al.(6) published the now classic paper on miniaturized total analysis systems (mu TAS). This resulted in a tremendous increase in the research of chip-based microfluidic systems, much like the revolution in the electronic industry after the invention of the transistor and the integrated circuit.

In the present study, effects of the alpha(2)- and beta-adrenoceptor agonists clonidine and isoproterenol on astrocytes in astroglial/neuronal cocultures from rat cerebral cortex were evaluated. The calcium- and potassium-sensitive dyes fura-2 and potassium-binding benzofuran isophtalate (PBFI) were used to study alterations in intracellular concentrations of calcium ([Ca(2+)](i)) and potassium ([K(+)](i)), respectively, while the perforated patch clamp technique was used to analyze transmembrane currents. Exposure to isoproterenol or clonidine elicited an immediate increase in [Ca(2+)](i) that was totally abolished in calcium-free extracellular media. Isoproterenol also decreased [K(+)](i), but clonidine did not. The reduction in [K(+)](i) was inhibited in Ca(2+)-free media. As evaluated with the perforated patch technique, isoproterenol (10(-6)-10(-4) M) induced a slowly developing and long lasting outward current that also was totally abolished in calcium-free buffer. This current was blocked by external tetraethylammonium (TEA, 10 mM) and charybdotoxin (ChTX, 10 nM), but was not affected by apamin (50 nM). The current-to-voltage (I-V) relationships for the isoproterenol-induced currents showed a markedly negative reversal potential, -96 mV+/-7, (mean+/-S.D., n=5). These results suggest that the stimulation of astroglial beta-adrenoceptors by isoproterenol opens calcium-activated potassium channels (K((Ca))). Preincubation with forskolin significantly increased the isoproterenol-induced currents compared with controls, indicating that the opening of astroglial K((Ca)) channels after beta-adrenergic stimulation not only depends on [Ca(2+)](i) but also synergistically involves the cAMP transduction system to which beta-adrenoceptors are known to be positively coupled.

Astroglia have the capacity to monitor extracellular glutamate (Glu) and maintain it at low levels, metabolize Glu, or release it back into the extracellular space. Glu can induce an increase in astroglial cell volume with a resulting decrease of the extracellular space, and thereby alter the concentration of extracellular substances. Many lines of evidence show that K+ can be buffered within the astroglial gap-junction-coupled network, and recent results show that gap junctions are permeable for Glu. All these events occur dynamically: the astroglial network has the capacity to interfere actively with neurotransmission, thereby contributing to a high signal-to-noise ratio for the Glu transmission. High-quality neuronal messages during normal physiology can then be maintained. With the same mechanisms, astroglia might exert a neuroprotective function in situations of moderately increased extracellular Glu concentrations, i.e., corresponding to conditions of pathological hyper-excitability, or corresponding to early stages of an acute brain injury. If the astroglial functions are failing, neuronal dysfunction can be reinforced.