Measuring Ca²âº changes in multiwell format using the Fluorometric Imaging Plate Reader (FLIPR(®)).

The Fluorometric Imaging Plate Reader (FLIPR) has made a significant contribution to drug discovery programs. The key advantage of FLIPR over conventional plate readers is the ability to measure fluorescence emission from multiple wells (96 wells or 384 wells) simultaneously and with high temporal resolution. Consequently, FLIPR has been used extensively to record dynamic intracellular processes such as changes in intracellular Ca(2+) ion concentration, membrane potential, and pH. Since FLIPR is used to measure a functional response in cells, it is rapidly able to distinguish full agonists, partial agonists, and antagonists at a target of interest, making the system a valuable screening tool for interrogation of compound libraries. Automated FLIPR systems for ultra high throughput have also become available that employ integrated plate stackers, washers and specialized stages to allow users to shuttle cell and compound plates from incubators or storage magazines onto the FLIPR system itself. In this chapter generic methods for assessing intracellular Ca(2+) on the FLIPR are described.

Culture of dissociated sensory neurons from dorsal root ganglia of postnatal and adult rats.

The development of new therapeutics for management of pain is likely to become much more mechanism based, and therefore, we need a more thorough understanding of the different pain development pathways. The afferent fibers of sensory neurons, with their cell bodies in the dorsal root ganglia (DRG), are thought to be key in pain mechanisms. DRG neurons can be prepared from embryonic, postnatal, or adult tissue. Embryonic preparations have the advantage of higher cell yields and greater proportion of neurons, but they are dependent on neurotrophins for the first week of culture. In contrast, dissociated postnatal and adult DRG sensory neurons offer the possibility to study mature neurons that may better resemble the in vivo characteristics of these cells. Here, we describe the dissociation of DRG sensory neurons from postnatal and adult rats. DRG are dissected and dissociated using a prolonged trypsin/collagenase treatment, followed by mechanical separation of the neurons. We have routinely prepared them for electrophysiological studies by the methods outlined in this chapter and describe some of the pitfalls that we have encountered, with hints of how to overcome them.

Abeta(1-42) reduces synapse number and inhibits neurite outgrowth in primary cortical and hippocampal neurons: a quantitative analysis.

Synaptic loss represents one of the earliest signs of neuronal damage and is observed within both Alzheimer's disease patients and transgenic mouse models of the disease. We have developed a novel in vitro assay using high content screening technology to measure changes in a number of cell physiological parameters simultaneously within a neuronal population. Using Hoechst-33342 to label nuclei, betaIII-tubulin as a neuron-specific marker, and synapsin-I as an indicator of pre-synaptic sites, we have designed software to interrogate triple-labelled images, counting only those synaptic puncta associated with tubulin-positive structures. Here we demonstrate that addition of amyloid beta peptide (Abeta(1-42)), to either primary hippocampal or cortical neurons for 4 days in vitro has deleterious effects upon synapse formation, neurite outgrowth and arborisation in a concentration-dependent manner. Control reverse peptide showed no effect over the same concentration range. The effects of Abeta(1-42) were inhibited by D-KLVFFA, which contains residues 16-20 of Abeta that function as a self-recognition element during Abeta assembly and bind to the homologous region of Abeta and block its oligomerisation. These effects of Abeta(1-42) on synapse number and neurite outgrowth are similar to those described within AD patient pathology and transgenic mouse models.

Activation of recombinant human TRPV1 receptors expressed in SH-SY5Y human neuroblastoma cells increases [Ca(2+)](i), initiates neurotransmitter release and promotes delayed cell death.

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.

TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia.

We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment. Activation of C13 cells by interleukin-1beta leads to a fivefold increase of TRPM2 mRNA demonstrating transcriptional regulation. To confirm mRNA distribution correlated with functional expression, we combined electrophysiology, Ca2+ imaging, and antisense approaches. C13 microglia exhibited, when stimulated with hydrogen peroxide (H2O2), increased [Ca2+]i, which was reduced by antisense treatment. Moreover, patch-clamp recordings from C13 demonstrated that increased intracellular adenosine diphosphoribose (ADPR) or extracellular H2O2 induced an inward current, consistent with activation of TRPM2. In addition we confirm the functional expression of a TRPM2-like conductance in primary microglial cultures derived from rats. Activation of TRPM2 in microglia during ischemic brain injury may mediate key aspects of microglial pathophysiological responses.

Measuring Ca²+ changes in multiwell format using the Fluorometric Imaging Plate Reader.

The Fluorometric Imaging Plate Reader (FLIPR®; Molecular Devices, Sunnyvale, CA) has made a significant contribution to drug discovery programs in the pharmaceutical industry since the first commercial instruments were introduced 9 yr ago. The key advantage of FLIPR over conventional plate readers is its ability to measure fluorescence emission from multiple wells (96- or 384-well) simultaneously and with high temporal resolution. Consequently, FLIPR has been used extensively to record dynamic intracellular processes such as changes in intracellular Ca(2+) ion concentration, membrane potential, and pH. Since FLIPR is used to measure a functional response in cells, it is rapidly able to distinguish full agonists, partial agonists, and antagonists at a target of interest, making the system a valuable screening tool for interrogation of compound libraries. Typically, FLIPR can be used to screen more than 150 compound plates per day in a high-throughput screening environment equating to more than 50,000 compounds at a single concentration in a 384-well system.

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores.

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.