Wayne Rasband of NIH has created ImageJ, an open source Java-written program that is now at version 1.31 and is used for many imaging applications, including those that that span the gamut from skin analysis to neuroscience. ImageJ is in the public domain and runs on any operating system (OS). ImageJ is easy to use and can do many imaging manipulations. A very large and knowledgeable group makes up the user community for ImageJ. Topics covered are imaging abilities; cross platform; image formats support as of June 2004; extensions, including macros and plug-ins; and imaging library. NIH reports tens of thousands of downloads at a rate of about 24,000 per month currently. ImageJ can read most of the widely used and significant formats used in biomedical images. Manipulations supported are read/write of image files and operations on separate pixels, image regions, entire images, and volumes (stacks in ImageJ). Basic operations supported include convolution, edge detection, Fourier transform, histogram and particle analyses, editing and color manipulation, and more advanced operations, as well as visualization. For assistance in using ImageJ, users e-mail each other, and the user base is highly knowledgeable and will answer requests on the mailing list. A thorough manual with many examples and illustrations has been written by Tony Collins of the Wright Cell Imaging Facility at Toronto Western Research Institute and is available, along with other listed resources, via the Web.

/pdf/image-processing-with-imagej-2nw6tuidbb.pdf

Image processing with ImageJ

Capsaicin, the main pungent ingredient in 'hot' chilli peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system We have used an expression cloning strategy based on calcium influx to isolate a functional cDNA encoding a capsaicin receptor from sensory neurons This receptor is a non-selective cation channel that is structurally related to members of the TRP family of ion channels The cloned capsaicin receptor is also activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo

/pdf/the-capsaicin-receptor-a-heat-activated-ion-channel-in-the-57mhjl9hkd.pdf

The capsaicin receptor: a heat-activated ion channel in the pain pathway

Protein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. When cells are stimulated, protein kinase C is transiently activated by diacylglycerol which is produced in the membrane during the signal-induced turnover of inositol phospholipids. Tumour-promoting phorbol esters, when intercalated into the cell membrane, may substitute for diacylglycerol and permanently activate protein kinase C. The enzyme probably serves as a receptor for the tumour promoters. Further exploration of the roles of this enzyme may provide clues for understanding the mechanism of cell growth and differentiation.

The role of protein kinase C in cell surface signal transduction and tumour promotion

Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

Inositol trisphosphate and calcium signalling

There has recently been rapid progress in understanding receptors that generate intracellular signals from inositol lipids. One of these lipids, phosphatidylinositol 4,5-bisphosphate, is hydrolysed to diacylglycerol and inositol trisphosphate as part of a signal transduction mechanism for controlling a variety of cellular processes including secretion, metabolism, phototransduction and cell proliferation. Diacylglycerol operates within the plane of the membrane to activate protein kinase C, whereas inositol trisphosphate is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium.

Inositol trisphosphate, a novel second messenger in cellular signal transduction.

The regulation of capacitative calcium entry by calcium and protein kinase C in Xenopus oocytes.

Neural stem cells can generate in vitro progenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes in these cells are poorly understood. Here we tested the hypothesis that Ca 2+ signaling controls differentiation of neural precursors. We found low-frequency global and local Ca 2+ transients occurring predominantly during early stages of differentiation. Spontaneous Ca 2+ signals in individual precursors were not synchronized with Ca 2+ transients in surrounding cells. Experimentally induced changes in the frequency of local Ca 2+ signals and global Ca 2+ rises correlated positively with neurite outgrowth and the onset of GABAergic neurotransmitter phenotype, respectively. NMDA receptor activity was critical for alterations in neuronal morphology but not for the timing of the acquisition of the neurotransmitter phenotype. Thus, spontaneous Ca 2+ signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency may specify neuronal morphology and acquisition of neurotransmitter phenotype.

https://www.jneurosci.org/content/jneuro/23/1/103.full.pdf

Local and Global Spontaneous Calcium Events Regulate Neurite Outgrowth and Onset of GABAergic Phenotype during Neural Precursor Differentiation

We examined the regulation of calcium signalling in atrial cardiomyocytes during excitation-contraction coupling, and how changes in the distribution of calcium impacts on contractility. Under control conditions, calcium transients originated in subsarcolemmal locations and showed local regeneration through activation of calcium-induced calcium release from ryanodine receptors. Despite functional ryanodine receptors being expressed at regular (∼2 μm) intervals throughout atrial myocytes, the subsarcolemmal calcium signal did not spread in a fully regenerative manner through the interior of a cell. Rather, there was a diminishing centripetal propagation of calcium. The lack of regeneration was due to mitochondria and SERCA pumps preventing the inward movement of calcium. Inhibiting these calcium buffering mechanisms allowed the globalisation of action potential-evoked responses. In addition, physiological positive inotropic agents, such as endothelin-1 and β-adrenergic agonists, as well as enhanced calcium current, calcium store loading and inositol 1,4,5-trisphosphate infusion also led to regenerative global responses. The consequence of globalising calcium signals was a significant increase in cellular contraction. These data indicate how calcium signals and their consequences are determined by the interplay of multiple subcellular calcium management systems.

/pdf/the-spatial-pattern-of-atrial-cardiomyocyte-calcium-38nzo2bnnt.pdf

The spatial pattern of atrial cardiomyocyte calcium signalling modulates contraction

Alzheimer's disease (AD) begins with a decline in cognition followed by neuronal cell death and dementia. These changes have been linked to a deregulation of Ca2+ signalling caused by a progressive increase in the resting level of Ca2+, which may influence cognition by interfering with the rhythm rheostat that controls the sleep/wake cycle. The rise in resting levels of Ca2+ may not alter the processes of memory acquisition during consciousness (gamma and theta rhythms), but may duplicate some of the events that occur during the slow oscillations responsible for the twin processes of memory consolidation and memory erasure that occur during sleep. The persistent elevation in the resting level of Ca2+ induced by an accumulation of amyloid β (Aβ) oligomers duplicates a similar small global elevation normally restricted to the period of slow oscillations when memories are erased during sleep. In AD, such a rapid erasure of memories soon after they are acquired during the wake period means that they are not retained for consolidation during sleep. The Aβ deregulates Ca2+ signalling through direct effects on the neurons and indirectly by inducing inflammatory responses in the microglia and astrocytes. Some of these deleterious effects of Aβ may be alleviated by vitamin D.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2013.257527

Calcium regulation of neural rhythms, memory and Alzheimer's disease

Excitation-contraction coupling (E-C coupling) was studied in isolated fluo-3-loaded rat atrial myocytes at 22 and 37 degrees C using rapid confocal microscopy. Within a few milliseconds of electrical excitation, spatially discrete subsarcolemmal Ca2+ signals were initiated. Twenty to forty milliseconds after stimulation the spatial overlap of these Ca2+ signals gave a 'ring' of elevated Ca2+ around the periphery of the cells. However, this ring was not continuous and substantial Ca2+ gradients were observed. The discrete subsarcolemmal Ca2+-release sites, which responded in a reproducible sequence to repetitive depolarisations and displayed the highest frequencies of spontaneous Ca2+ sparks in resting cells, were denoted 'eager sites'. Immunostaining atrial myocytes for type II ryanodine receptors (RyRs) revealed both subsarcolemmal 'junctional' RyRs, and also 'non-junctional' RyRs in the central bulk of the cells. A subset of the junctional RyRs comprises the eager sites. For cells paced in the presence of 1 mM extracellular Ca2+, the response was largely restricted to a subsarcolemmal 'ring', while the central bulk of the cell displayed a approximately 5-fold lower Ca2+ signal. Under these conditions the non-junctional RyRs were only weakly activated during E-C coupling. However, these channels are functional and the Ca2+ stores were at least partially loaded, since substantial homogeneous Ca2+ signals could be stimulated in the central regions of atrial myocytes by application of 2.5 mM caffeine. Neither the location nor activation order of the eager sites was affected by increasing the trigger Ca2+ current (by increasing extracellular Ca2+ to 10 mM) or the sarcoplasmic reticulum (SR) Ca2+ load (following 1 min incubation in 10 mM extracellular Ca2+), although with increased SR Ca2+ load, but not greater Ca2+ influx, the delay between the sequential activation of eager sites was reduced. In addition, increasing the trigger Ca2+ current or the SR Ca2+ load changed the spatial pattern of the Ca2+ response, in that the Ca2+ signal propagated more reliably from the subsarcolemmal initiation sites into the centre of the cell. Due to the greater spatial spread of the Ca2+ signals, the averaged global Ca2+ transients increased by approximately 500 %. We conclude that rat atrial myocytes display a predetermined spatiotemporal pattern of Ca2+ signalling during early E-C coupling. A consistent set of eager Ca2+ release sites with a fixed location and activation order on the junctional SR serve to initiate the cellular response. The short latency for activation of these eager sites suggests that they reflect clusters of RyRs closely coupled to voltage-operated Ca2+ channels in the sarcolemma. Furthermore, their propensity to show spontaneous Ca2+ sparks is consistent with an intrinsically higher sensitivity to Ca2+-induced Ca2+ release. While the subsarcolemmal Ca2+ response can be considered as stereotypic, the central bulk of the cell grades its response in direct proportion to cellular Ca2+ load and Ca2+ influx.

Michael J. Berridge

Papers

The regulation of capacitative calcium entry by calcium and protein kinase C in Xenopus oocytes.

Local and Global Spontaneous Calcium Events Regulate Neurite Outgrowth and Onset of GABAergic Phenotype during Neural Precursor Differentiation

The spatial pattern of atrial cardiomyocyte calcium signalling modulates contraction

Calcium regulation of neural rhythms, memory and Alzheimer's disease

Predetermined recruitment of calcium release sites underlies excitation-contraction coupling in rat atrial myocytes.