Calcium Indicator Dye Research Articles

Endothelin-1–Induced Proliferation Is Reduced and Ca2+Signaling Is Enhanced in Endothelin B–Deficient Optic Nerve Head Astrocytes

To characterize the influence of endothelin-1 (ET-1) on optic nerve head astrocyte (ONHA) proliferation and Ca²⁺ signaling in ONHAs lacking functional endothelin B (ETB) receptors. ONHAs were isolated from adult wild type (WT) and transgenic spotting lethal (TSL) rats, lacking functional ETB receptors. ONHA specificity was confirmed by positive glial fibrillary acidic protein (GFAP), negative A2B5 (a marker for type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP) labeling. The mitogenic effects of 10⁻⁷ or 10⁻⁹ M ET-1, or vehicle were investigated for 48 or 72 hours in WT and TSL ONHAs. Intracellular calcium levels ([Ca²⁺](i)) were assessed in ONHAs loaded with fura-2 calcium indicator dye. ET-1-induced proliferation of TSL ONHAs was blunted at 48 hours (by 37% at 10⁻⁷ M and by 33% at 10⁻⁹ M) and 72 hours (by 117% at 10⁻⁷ M and by 100% at 10⁻⁹ M) compared with WT cells. ET-1-induced ONHA fura-2 ratio increases were significantly greater in TSL ONHAs (by 20% at 10⁻⁷ M and by 48% at 10⁻⁹ M) compared with WT ONHAs. ET-1-induced fura-2 ratio increases were blocked after pretreatment with BQ-610 (ETA antagonist) in WT and TSL ONHAs, but not by BQ-788 (ETB antagonist) in WT ONHAs. ET-1-induced ONHA proliferation is reduced in cells lacking functional ETB receptors, ET-1-induced [Ca²⁺](i) increases are enhanced in the absence of functional ETB receptors, and ETA, but not ETB, is required for ET-1-induced [Ca²⁺](i) elevation.

1 A ROLE FOR CHEMOKINES AND NGF IN PAIN GENERATION IN A MURINE MODEL OF OSTEOARTHRITIS

Purpose: Pain is the major symptom in osteoarthritis (OA), yet how this pain develops remains unknown. We recently reported that destabilization of the medial meniscus (DMM) provides a suitable mouse model for studying pain development and progression at different stages of OA. We found that, following DMM in the right knee, male C57BL/6 mice develop progressive mechanical allodynia in the ipsilateral hindpaw, as early as 2 weeks and progressing over 8 weeks post DMM (but not sham) surgery. Additionally, total distance traveled over a specified period was relatively constant over the first 8 weeks, but decreased from 12 to 16 weeks after surgery. The current goal was to identify molecular pathways in the knee joint and its innervating nociceptive system that correlate with these different stages of disease. Methods: DMM or sham surgery was performed in the right knee of 10-week old male C57BL/6 mice. At 8 and 16 weeks post surgery, histopathology of the knees was evaluated according to OARSI recommendations. Additionally, at 4 and 8 weeks after surgery, whole knee joints were collected for protein extraction and ELISA. At 4, 8, or 16 weeks post surgery, innervating dorsal root ganglia (DRG), L3L5, from DMM or sham-operated mice and age-matched naive mice were collected for protein extraction for ELISA or for mRNA extraction for quantitative RT-PCR of nerve growth factor (NGF), monocyte chemoattractant protein (MCP)-1 and its receptor CCR2, and stromal cell-derived factor (SDF)-1. Finally, at 8 weeks post surgery, the response of DRG neurons to chemokines was recorded though intracellular Ca2+imaging, following standard protocols. In brief, DRG neurons were acutely isolated, plated on coverslips, cultured for 3–4 days, and loaded with a calcium indicator dye. The number of cells responding to chemokines was counted. Results: Following DMM (but not sham) surgery, C57BL/6 mice developed cartilage degeneration and osteophyte formation by 8 weeks in the ipsilateral knee only, with progressive cartilage deterioration occurring up to 16 weeks. At 4 weeks post DMM, protein levels of MCP-1 and NGF were elevated in total joint extracts from the operated knee compared to naive age-matched controls (p = 0.025 and p=0.099, respectively); by 8 weeks post DMM, levels of both proteins had returned to baseline. mRNA levels of NGF, MCP-1, CCR2, and SDF-1 in the innervating DRGwere upregulated compared to naive and sham age-matched controls, peaking at 8 weeks post surgery. Protein levels of NGF were also increased in DRG at 8 weeks post surgery (p = 0.03), while MCP-1 protein was below the detection limit at all time points. Exposing isolated DRG neurons from DMM mice at 8 weeks post surgery to MCP-1 or to SDF-1 (200 or 500ng/mL, respectively) resulted in an increased intracellular calcium response compared to naive age-matched controls, indicating an upregulation of their respective receptors, CCR2 and CXCR4, on these cells. Conclusions: In this study, gene and protein expression in the knee joint and innervating DRG of DMM mice reveal temporal changes in NGF, SDF-1 and MCP-1 during the development of painful OA. In addition, Ca-imaging studies suggest a functional role for the chemokines MCP-1 and SDF-1 in pain generation in this model. NGF is known to play a role in sensitization of the nervous system in inflammation-associated pain; recent research has shown that the chemokines MCP-1 and SDF-1 contribute to the development of chronic pain in neuropathic pain models. Therefore, this work supports burgeoning clinical evidence that OA-related pain may include aspects of both inflammatory and neuropathic pain.

Heuristically optimal path scanning for high-speed multiphoton circuit imaging

Population dynamics of patterned neuronal firing are fundamental to information processing in the brain. Multiphoton microscopy in combination with calcium indicator dyes allows circuit dynamics to be imaged with single-neuron resolution. However, the temporal resolution of fluorescent measures is constrained by the imaging frequency imposed by standard raster scanning techniques. As a result, traditional raster scans limit the ability to detect the relative timing of action potentials in the imaged neuronal population. To maximize the speed of fluorescence measures from large populations of neurons using a standard multiphoton laser scanning microscope (MPLSM) setup, we have developed heuristically optimal path scanning (HOPS). HOPS optimizes the laser travel path length, and thus the temporal resolution of neuronal fluorescent measures, using standard galvanometer scan mirrors. Minimizing the scan path alone is insufficient for prolonged high-speed imaging of neuronal populations. Path stability and the signal-to-noise ratio become increasingly important factors as scan rates increase. HOPS addresses this by characterizing the scan mirror galvanometers to achieve prolonged path stability. In addition, the neuronal dwell time is optimized to sharpen the detection of action potentials while maximizing scan rate. The combination of shortest path calculation and minimization of mirror positioning time allows us to optically monitor a population of neurons in a field of view at high rates with single-spike resolution, ∼ 125 Hz for 50 neurons and ∼ 8.5 Hz for 1,000 neurons. Our approach introduces an accessible method for rapid imaging of large neuronal populations using traditional MPLSMs, facilitating new insights into neuronal circuit dynamics.

Bulk Loading of Calcium Indicator Dyes to Study Astrocyte Physiology: Key Limitations and Improvements Using Morphological Maps

Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ∼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as "maps" after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.

Population calcium imaging of spontaneous respiratory and novel motor activity in the facial nucleus and ventral brainstem in newborn mice

The brainstem contains rhythm and pattern forming circuits, which drive cranial and spinal motor pools to produce respiratory and other motor patterns. Here we used calcium imaging combined with nerve recordings in newborn mice to reveal spontaneous population activity in the ventral brainstem and in the facial nucleus. In Fluo-8AM loaded brainstem-spinal cord preparations, respiratory activity on cervical nerves was synchronized with calcium signals at the ventrolateral brainstem surface. Individual ventrolateral neurons at the level of the parafacial respiratory group showed perfect or partial synchrony with respiratory nerve bursts. In brainstem-spinal cord preparations, cut at the level of the mid-facial nucleus, calcium signals were recorded in the dorsal, lateral and medial facial subnuclei during respiratory activity. Strong activity initiated in the dorsal subnucleus, followed by activity in lateral and medial subnuclei. Whole-cell recordings from facial motoneurons showed weak respiratory drives, and electrical field potential recordings confirmed respiratory drive to particularly the dorsal and lateral subnuclei. Putative facial premotoneurons showed respiratory-related calcium signals, and were predominantly located dorsomedial to the facial nucleus. A novel motor activity on facial, cervical and thoracic nerves was synchronized with calcium signals at the ventromedial brainstem extending from the level of the facial nucleus to the medulla–spinal cord border. Cervical dorsal root stimulation induced similar ventromedial activity. The medial facial subnucleus showed calcium signals synchronized with this novel motor activity on cervical nerves, and cervical dorsal root stimulation induced similar medial facial subnucleus activity. In conclusion, the dorsal and lateral facial subnuclei are strongly respiratory-modulated, and the brainstem contains a novel pattern forming circuit that drives the medial facial subnucleus and cervical motor pools.

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

Frequency Induced Alterations in Calcium Signaling

The Bowditch effect, otherwise known as the force frequency response, is a major endogenous physiological regulator of the contractile function of the heart where an increase in pacing frequency leads to an increase in force. This is critical in exercise, where the increase in activity elicits an increase in the delivery of oxygenated blood to the rest of the body. Previous studies in our lab have shown that at steady state, higher frequencies lead to a decrease in calcium transient duration and alterations in the phosphorylation status of myofilament proteins. In this study we aim to determine the regulatory modifications that occur in contractile function, protein phosphorylation, and calcium handling, during the transition in the intact cardiac muscle. Trabeculae, excised from the right ventricular free wall of New Zealand white rabbits, are suspended in Krebs-Hensleit solution and stimulated at 1Hz. Muscles were then incubated at room temperature with Fluo-4 am calcium indicator dye at50uM for 30-60 minutes. Dye loaded muscles were then stimulated to contract at 1 Hz followed by 4 Hz over the course of 4 minutes. Increasing the frequency from 1 to 4 Hz leads to an alteration in contractile function with a transition phase occurring between steady-states. Functional data reveals that the transition phase is characterized by a fast response, in which there is a transient decrease followed by a rapid increase in contractile force, this is then proceeded by a slow response, in which the contraction gradually increases in force until reaching a steady-state at 4 Hz. Currently, we are obtaining calcium transient data from the transition phase to correlate the effects of increasing frequency, force, and calcium flux. Further studies are required to determine the effects of frequency on post-translational modifications at these transitional phases.

Spontaneous Calcium Changes in Micro Neuronal Networks

We have developed a practical experimental method to mass-produce and maintain a variation of minimal neuronal networks (“micro neuronal networks”) consisted of a single to several neurons in culture using spray-patterning technique. In this paper, we could maintain the micro-cultures for one month or more by adding conditioned medium and carried out optical recording of spontaneous activity in micro neuronal networks and considered the interactions between them. To determine the interactions between micro neuronal networks, fluorescence changes in several small networks were simultaneously measured using calcium indicator dye fluo-4 AM, and time-series analysis was carried out using surrogate arrangements. By using the spray-patterning method, a large number of cell-adhesive micro regions were formed. Neurons extended neurites along the edge of the cell-adhesive micro regions and form micro neuronal networks. In part of micro regions, some neurite was protruded from the region, and thus micro neuronal networks were connected with synapses. In these networks, a single neuron-induced network activity was observed. On the other hand, even in morphologically non-connected micro neuronal networks, synchronous oscillations between micro neuronal networks were observed. Our micro-patterning methods and results provide the possibility that synchronous activity is occurred between morphologically non-connected neuronal networks. This suggest that the humoral factor is also a important component for network-wide dynamics.

Visualizing Calcium Signaling in Astrocytes

Astrocytes are nonneuronal cells in the brain (glia) that do not generate electrical impulses but communicate by chemical signaling. This communication can be observed under a microscope with fluorescent calcium indicators that glow more brightly when the concentration of calcium increases inside the cell. Astrocytes release adenosine 5'-triphosphate and other cell signaling molecules that excite membrane receptors on other astrocytes to cause an increase in intracellular calcium in the recipient cell. Many of the substances released by astrocytes also excite neurons, and astrocytes have on their own cell membrane many of the same neurotransmitter receptors used by neurons to communicate across synapses. This allows astrocytes to respond to neural impulse activity, communicate among other astrocytes, and influence neuronal communication by taking up or releasing neurotransmitters from synapses.

Antidepressants act on glial cells: SSRIs and serotonin elicit astrocyte calcium signaling in the mouse prefrontal cortex

One important target in the treatment of major depressive disorder (MDD) is the serotonin (5-hydroxytryptamine, 5-HT) system. Selective serotonin reuptake inhibitors (SSRI) are used to treat MDD. Yet, the mode of action of these drugs is not completely understood. There is evolving evidence for a role of glutamate in mood disorder and its signaling. Astrocytes are involved in glutamate metabolism and play an active role in memory processing but their role in mood disorders is still largely unknown. A modulation of astrocytic signaling by SSRIs or 5-HT has not been investigated up to now. We investigated astrocytic calcium signaling with the calcium indicator dye Fluo-4. Using a confocal microscope, we imaged astrocytes in the medial prefrontal cortex of acute mouse brain slices after the application of the SSRIs citalopram and fluoxetine. In the same way, we studied the effects of serotonin and the modulation of this signaling by glutamate in astrocytes. We found that astrocyte calcium signaling can be elicited by 5-HT. Also, the SSRIs citalopram and fluoxetine induce calcium signals in about 1/3 of all astrocytes, even when neuronal signal propagation is inhibited. Astrocytic responses to 5-HT have a unique pattern and they could mostly not be evoked twice. We determined that glutamate is a substance that can interfere with 5-HT-induced calcium signals in astrocytes since after stimulation by glutamate, astrocytes did not show a response to 5-HT. Astrocytic calcium signaling is elicited by SSRIs and 5-HT. They may serve as integrators, linking the serotonergic and glutamatergic signaling pathways.

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Since Cajal's early drawings, the characterization of neuronal architecture has been paramount in understanding neuronal function. With the development of electrophysiological techniques that provide unprecedented access to the physiology of these cells, experimental questions of neuronal function have also become more tractable. Fluorescent tracers that can label the anatomy of individual or populations of neurons have opened the door to linking anatomy with physiology. Experimentally however, current techniques for bulk labeling of cells in vitro often affect neuronal function creating a barrier for exploring structure-function questions. Here we describe a new technique for highly localized electroporation within a cell or cell population that enables the introduction of membrane impermeable charged dyes including dextran-conjugated fluorophores, hydrazide tracers, and calcium indicator dyes in vitro. We demonstrate that this technique is highly versatile, allowing for labeling of large or small areas of tissue, allowing for the investigation of both cellular morphology and physiological activity in identified neuronal circuits in acute brain slices. Furthermore, this approach allows subsequent targeted whole-cell patch recording based on well-defined connectivity as well as assessment of physiological activity in targeted circuits on a fast time scale.

Clozapine activates AMP-activated protein kinase (AMPK) in C2C12 myotube cells and stimulates glucose uptake

Aims Clozapine has previously been implicated in the dysregulation of energy balance and glucose metabolism in the central nervous system, but its effects in the periphery have yet to be thoroughly elucidated. The objective of this study was to characterize the effects of clozapine on AMP-activated protein kinase (AMPK) activity in the skeletal muscles. Main methods Myotube C2C12 cells were incubated under control conditions, or with clozapine. Expression levels of phosphorylation status of AMPK and its direct downstream Acetyl-CoA carboxylase (ACC) were analyzed by Western blot. Intracellular calcium concentration was measured with calcium indicator dye, fluo-3 AM. 2-deoxyglucose uptake was assessed via the scintillation count. Key findings We reported that clozapine activated AMPK in mouse C2C12 myotubes and also stimulated glucose uptake. Clozapine also increased intracellular calcium concentrations of C2C12 cells, and pretreatment with either ethylenediaminetetraacetic acid (EDTA), an extracellular calcium chelator, or 1.8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), a Ca 2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, blocked clozapine-induced AMPK activation. Significance These results demonstrate that clozapine increases glucose uptake through CaMKK-AMPK pathway in myotube C2C12 cells.

Abstract 4028: A consolidated microplate-based workflow for monitoring intracellular calcium mobilization and mitochondrial membrane potential changes

Abstract The interactions between mitochondrial function and Ca2+ homeostasis are very complex. Mitochondria are known to serve as dynamic calcium storage compartments, and also form intimate contacts with other calcium-handling organelles, such as the endoplasmic reticulum. Calcium enters mitochondria, not only during pathological processes, but also during physiological calcium responses. This organelle does not simply passively buffer excessive calcium but actively utilizes calcium signals to regulate integral cell activities, such as oxidative phosphorylation and the Kreb's cycle. A consolidated microplate-based workflow is described for assessing the relationship between mitochondria and global calcium responses. Two novel small molecule probes were implemented in the assay. Mitochondrial membrane potential was monitored using a dual-emission fluorescent probe that is roughly 10-25-fold more sensitive than the conventional carbocyanine dye, JC-1. Calcium mobilization was examined using a novel fluorescent calcium indicator dye that is significantly brighter than Fluo-4 AM dye. The assay is readily performed using a fluorescence microplate reader equipped with a dual-reagent dispenser. Proof-of-principle studies were performed by evaluating cell response to two agents, diazoxide, a putative opener of the potassium-ATP channel, and BML-258, a novel sphingosine kinase-1 inhibitor. The assay workflow is suitable for monitoring calcium mobilization and mitochondrial membrane potential changes across a broad spectrum of biological targets. The easy-to-use protocol is automation-friendly and can be performed in a convenient 96-well or 384-well microplate format. The assay should provide insight into the interaction between calcium signaling and mitochondrial energetic status, relevant to a range of phenomena, including ischemia/reperfusion injury. It should also serve as a high-throughput approach for identifying specific small molecule pharmacological agents targeted to this complex interrelationship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4028.

C-peptide stimulates nitrites generation via the calcium-JAK2/STAT1 pathway in murine macrophage Raw264.7 cells

Aims C-peptide is a product of pro-insulin cleavage. Numerous studies have demonstrated that C-peptide, although not influencing blood glucose control, may play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. The aim of this paper was to present a novel function of C-peptide, focusing on its role in nitric oxide (NO) generation. Main methods Murine macrophage Raw264.7 cells and primary peritoneal macrophages were incubated under control conditions, or with C-peptide. Expression level of iNOS and phosphorylation status of JAK2/STAT1 were analyzed by Western blot. Fluorometric NO assay kit was used to assess the concentration of nitrite in culture medium. Intracellular calcium concentration was measured with calcium indicator dyes, such as Fura-2 and Fluo-3 AM. Key findings C-peptide increased the level of nitrites in murine macrophage Raw264.7 cells. The nitrites production induced by lipopolysaccharide (LPS) was further enhanced by co-treatment of C-peptide. This up-regulation of nitrites generation also correlated with the induction of inducible nitric oxide synthase (iNOS), a prominent marker of macrophage activation. In addition, C-peptide increased the intracellular concentration of calcium levels. Moreover, C-peptide-induced nitrites generation and increase in calcium was observed in freshly isolated primary peritoneal macrophages. In addition, C-peptide specifically affected the Janus activated kinase (JAK)/signal transducer and activated transcription (STAT) pathway. Finally, C-peptide-mediated nitrites generation and JAK2/STAT1 phosphorylation were not detected in the presence of the intracellular calcium chelator, BAPTA-AM. Significance These results suggest that C-peptide may elicit immune modulatory function via modulation of the calcium/JAK–STAT pathway.

Microfluidic Local Perfusion Chambers for the Visualization and Manipulation of Synapses

The polarized nature of neurons and the size and density of synapses complicates the manipulation and visualization of cell biological processes that control synaptic function. Here we developed a microfluidic local perfusion (microLP) chamber to access and manipulate synaptic regions and presynaptic and postsynaptic compartments in vitro. This chamber directs the formation of synapses in >100 parallel rows connecting separate neuron populations. A perfusion channel transects the parallel rows, allowing access with high spatial and temporal resolution to synaptic regions. We used this chamber to investigate synapse-to-nucleus signaling. Using the calcium indicator dye Fluo-4 NW, we measured changes in calcium at dendrites and somata, following local perfusion of glutamate. Exploiting the high temporal resolution of the chamber, we exposed synapses to "spaced" or "massed" application of glutamate and then examined levels of pCREB in somata. Lastly, we applied the metabotropic receptor agonist DHPG to dendrites and observed increases in Arc transcription and Arc transcript localization.

The 2009 Lindau Nobel Laureate Meeting: Roger Y. Tsien, Chemistry 2008

American biochemist Roger Tsien shared the 2008 Nobel Prize in Chemistry with Martin Chalfie and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Tsien, who was born in New York in 1952 and grew up in Livingston New Jersey, began to experiment in the basement of the family home at a young age. From growing silica gardens of colorful crystallized metal salts to attempting to synthesize aspirin, these early experiments fueled what would become Tsien's lifelong interest in chemistry and colors. Tsien's first official laboratory experience was an NSF-supported summer research program in which he used infrared spectroscopy to examine how metals bind to thiocyanate, for which he was awarded a $10,000 scholarship in the Westinghouse Science Talent Search. Following graduation from Harvard in 1972, Tsien attended Cambridge University in England under a Marshall Scholarship. There he learned organic chemistry --a subject he'd hated as an undergraduate-- and looked for a way to synthesize dyes for imaging neuronal activity, generating BAPTA based optical calcium indicator dyes. Following the completion of his postdoctoral training at Cambridge in 1982, Tsien accepted a faculty position at the University of California, Berkeley. There he and colleagues developed and improved numerous small molecule indicators, including indicators fura-2 and indo-1. In 1989, Tsien moved his laboratory to the University of California at San Diego, where he and his colleagues developed the enhanced mutant of GFP as a way to devise a cyclic AMP (cAMP) sensor for use in live cells. They initially engineered molecules to take advantage of the conformational change that occurs when cAMP binds to protein kinase A (PKA). By labeling one part of PKA with fluoroscein and another with a rhodamine, they hoped to detect Fluorescence Resonance Energy Transfer (FRET), which would occur when the two molecules were in close proximity. The initial experiments presented numerous difficulties due to the challenges of expressing PKA subunits in E. coli, labeling the protein without destroying its function, and delivering the protein to cells via microinjection. Eventually, Tsien sought a more elegant approach, hoping to use and modify a naturally fluorescent protein that could be expressed in the cell. GFP originally described by Davenport in 1955, extracted and purified by Shimomura in 1965, and cloned by Prasher in 1992 was an appealing candidate. To make the protein more useful for their FRET studies, Tsien and colleagues modified the amino acid structure of the protein (S65T). The improved protein had an excitation peak near that of fluoroscein, and was photostable. Tsien and colleagues also solved the protein's crystal structure, enabling them to generate additional colors with spectral properties suitable for FRET. However, when they attempted to use the GFP proteins in the detection of cAMP, they experienced further difficulties with PKA. Instead, their first successful use of GFP derivatives for FRET was in the detection of intracellular calcium using their engineered calmodulin-based calcium indicator, Cameleon. In a short time, Tsien's work has led to further technological developments and important scientific findings. GFP and its derivatives have been used in a wide range of biological applications, from the study of protein localization to understanding how HIV spreads from cell to cell. The need for such probes is highlighted by the abundance of research conducted using these fluorescent proteins, as well as the continued development of similar fluorescent proteins, such as the coral-derived dsRED. Tsien is currently developing genetically encoded Infrared Fluorescent Proteins (IFPs), which with their long emission wavelengths of >700 nm, have the ability to pass through living tissue and improve imaging in living organisms. He is also building synthetic molecules for use in humans. He cites team effort and the contributions of students and post-docs as key components of progress and success: "Even if I had the time, I couldn't have done the experiments, because I don't know how. It's very much a team effort."

Effects of a Y522S-RyR1 Mutation on Cerebellar Purkinje Cell Function

The devastating consequences of calcium dysregulation are exemplified by the skeletal muscle diseases malignant hyperthermia (MH) and central core disease (CCD). These diseases result from mutations in the ‘skeletal muscle-isoform’ of the ryanodine receptor calcium release channel (RyR1). To investigate the etiology of MH and CCD, mouse models have recently been generated (Chelu et al. 2005). Skeletal muscle harboring the Y522S-RyR1 knock-in mutation exhibit Ca2+ leak from internal stores, basal cellular stress, and ultimately progressive mitochondrial and cellular damage (Durham et al. 2008). Although the effects of this MH-causing mutation have been characterized in skeletal muscle, the effects in the nervous system have not been documented. RyR1 is expressed highly in cerebellar Purkinje neurons. Y522S-RyR1 mice do not exhibit gross neurological defects suggesting that the defects may be subtle or that neuronal function is spared due to compensatory changes.We have initiated studies investigating 3 main aspects of Y522S-RyR1 mouse Purkinje cells: intracellular calcium release, cellular organization/morphology, and cellular stress. In preliminary studies using imaging techniques with the calcium indicator dye Fura2-AM, RyR-mediated calcium release in Y522S-RyR1 Purkinje neurons exhibited a negative shift in the apparent EC50 for the agonist caffeine. In addition, the sensitivity of RyR to other activators (temperature, voltage, ryanodine) and intracellular calcium store content were investigated. Since calcium signaling is important during neuronal development and maturation, the morphology of Y522S-RyR1 Purkinje cells was examined using immunohistochemistry and confocal microscopy. Moreover, we have initiated studies to determine if altered RyR1 function results in basal cellular stress using Western blot and immunohistochemical analyses. Y522S-RyR1 mutant mice provide an excellent tool to address calcium dysregulation, its pathological consequences, and potential approaches for compensation of altered calcium signaling in the central nervous system.

Calcium Signaling in Neuronal Cells Exposed to the Munitions Compound Cyclotrimethylenetrinitramine (RDX)

Cyclotrimethylenetrinitramine (RDX) has been used extensively as an explosive in military munitions. Mechanisms for seizure production, seen in past animal studies, have not been described. Increased calcium levels contribute to excitotoxicity, so in this study neuroblastoma cells are loaded with calcium-indicating dye before application of 1.5 microM to 7.5 mM RDX, with fluorescence recorded for 30 cycles of 11 seconds each. The lowest concentration of RDX increases calcium fluorescence significantly above baseline for cycles 2 to 8; millimolar concentrations increase calcium fluorescence significantly above baseline for cycles 2 to 30. Increases in calcium, like those of 200 nM carbachol, are prevented with 10 mM of calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N tetra-acetic acid (EGTA, tetrasodium salt). Calcium channel blocker verapamil (20 microM), Ca(2+)-ATPase inhibitor thapsigargin (5 microM), and general membrane stabilizer lidocaine (10 mM) partially attenuate carbachol- and RDX-induced increases in calcium, suggesting that RDX transiently increases intracellular calcium by multiple mechanisms.

Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls

As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3 μm thick and spaced 10 μm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electropration, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.