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In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca$^{2+}$-dependent exocytosis regulated by distinct Ca$^{2+}$ sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca$^{2+}$ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

The 25-century long history of brain science can be divided into four distinct 'Eras': Era 1 (∼2000 years) started around 500 BC with reductionist studies of human and animal brains using invasive in vivo and ex vivo methods; Era 2 (∼200 years) started in the 17th century and introduced the first invasive methods to study the function of living tissues; Era 3 started around 1838 and is the ongoing era of cellular neurophysiology; finally, Era 4 (in statu nascendi) is the era of non-invasive, holistic yet mechanistic, studies of the brain. Animal experimentation is becoming increasingly more holistic as multimodal imaging and recording techniques are combined in a single experiment on the brain of awake behaving animal. This newly emerged approach can be called in vigilo (from Latin 'in awake' or 'in vigilant state'), by analogy to the earlier introduced terms in vivo or in vitro. We introduce the Special Issue "In Vigilo Veritas: New Frontiers of Optical Imaging and Electrical Recording in the Brain of Awake Behaving Mice", which features original research articles and reviews that represent some of the finest examples of a truly multimodal studies, where behavioural readouts and tasks are combined in the same longitudinal experiment and on the same mouse with the two-photon imaging, optogenetics and/or electrophysiological recordings. This exciting multi-methodological approach creates a fertile ground for breakthrough discoveries in neurophysiology and neuropsychology of an awake behaving mammalian brain.

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are morpho-functional units, formed at the loci of close apposition of the ER-forming endomembrane and outer mitochondrial membrane (OMM). These sites contribute to fundamental cellular processes including lipid biosynthesis, autophagy, apoptosis, ER-stress and calcium (Ca

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na

Astrocytes are the principal homeostatic cells of the central nerves system (CNS) that support the CNS function at all levels of organisation, from molecular to organ. Several fundamental homeostatic functions of astrocytes are mediated through plasmalemmal pumps and transporters; most of which are also regulated by the transplasmalemmal gradient of Na

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca

Day/night cycle controls neurogenesis; melatonin released from pineal gland in darkness stimulates intracellular Ca2+ dynamics thus decreasing proliferation of neural stem cells. In the daylight intracellular Ca2+ activity subsides, which stimulates neural stem cells division and increases generation of newborn neurones.

Graphical abstract Trace (A) shows a typical transient [Ca2+]i response induced by 50 mM K+ observed in an AVP-eGFP neurone (inset), Trace (B) shows the spontaneous [Ca2+]i oscillations observed in an AVP-eGFP neurone (inset). The corresponding changes in the fluorescence are shown in C and D, respectively., Highlights • Supraoptic fluorescent vasopressin (AVP-eGFP) and oxytocin (OT-mRFP1) neurones exhibit distinct spontaneous [Ca2+]i oscillations. • Vasopressin triggers [Ca2+]i oscillations, intensifies existing oscillations, and exceptionally stops oscillations. • Hyper- or hypo-osmotic stimuli have an intensifying or inhibitory effect on oscillations, respectively. • Nearly 90% of neurones from 3 or 5-day-dehydrated rats exhibit oscillations. • More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats are oscillatory vs. about 44% in virgins., The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca2+ signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca2+]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca2+]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca2+]i dynamics. In rats dehydrated for 3 or 5 days almost 90% of neurones displayed spontaneous [Ca2+]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca2+ signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.

International audience; Stem cells (SCs) of different origins have brought hope as potential tools for the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Calcium signalling plays a key role in SC differentiation and proliferation, and dysregulation of Ca(2+) homeostasis may instigate pathological scenarios. Currently, the role of ion channels and receptors in SCs is not fully understood. In the recent years, we found that (i) the pre-differentiation of human embryonic SCs (hESCs) led to the activation of Ca(2+) signalling cascades and enhanced the functional activities of these cells, (ii) the Ca(2+) homeostasis and the physiological properties of hESC-derived neural precursors (NPs) changed during long term propagation in vitro, (iii) differentiation of NPs derived from human induced pluripotent SCs affects the expression of ion channels and receptors, (iv) these neuronal precursors exhibited spontaneous activity, indicating that their electrophysiological and Ca(2+) handling properties are similar to those of mature neurones, and (v) in mesenchymal SCs isolated from the adipose tissue and bone marrow of rats the expression profile of ion channels and receptors depends not only on the differentiation conditions but also on the source from which the cells were isolated, indicating that the fate and functional properties of the differentiated cells are driven by intrinsic mechanisms. Together, identification and assignment of a unique ion channel and a Ca(2+) handling footprint for each cell type would be necessary to qualify them as physiologically suitable for medical research, drug screening, and cell therapy.

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na+, Ca2+) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca2+ at the expense of the Na+ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca2+ extrusion and thus to the recovery from intracellular Ca2+ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na+ to 1 Ca2+, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na+ concentration or minor depolarisations switch it into the “reverse mode” (Ca2+ import/Na+ export). Notably, transient Na+ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na+-permeable ion channels in response to neuronal activity. Activity-related Na+ transients result in NCX reversal, which mediates Ca2+ influx from the extracellular space, thereby generating astrocyte Ca2+ signalling independent from InsP3-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca2+ overload, while dampening Na+ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na+-driven, reverse NCX-mediated astrocyte Ca2+ signals are involved neurone-glia interaction. Na+ transients, translated by the NCX into Ca2+ elevations, thereby emerge as a new signalling pathway in astrocytes.

The aim of the present article is to analyse the evolutionary links between protozoa and neuronal and neurosecretory cells. To this effect we employ functional and topological data available for ciliates, in particular for Paramecium. Of note, much less data are available for choanoflagellates, the progenitors of metazoans, which currently are in the focus of metazoan genomic data mining. Key molecular players are found from the base to the highest levels of eukaryote evolution, including neurones and neurosecretory cells. Several common fundamental mechanisms, such as SNARE proteins and assembly of exocytosis sites, GTPases, Ca2+-sensors, voltage-gated Ca2+-influx channels and their inhibition by the forming Ca2+/calmodulin complex are conserved, albeit with different subcellular channel localisation, from protozoans to man. Similarly, Ca2+-release channels represented by InsP3 receptors and putative precursors of ryanodine receptors, which all emerged in protozoa, serve for focal intracellular Ca2+ signalling from ciliates to mammalian neuronal cells, eventually in conjunction with store-operated Ca2+-influx. Restriction of Ca2+ signals by high capacity/low affinity Ca2+-binding proteins is maintained throughout the evolutionary tree although the proteins involved differ between the taxa. Phosphatase 2B/calcineurin appears to be involved in signalling and in membrane recycling throughout evolution. Most impressive example of evolutionary conservation is the sub-second dynamics of exocytosis-endocytosis coupling in Paramecium cells, with similar kinetics in neuronal and neurosecretory systems. Numerous cell surface receptors and channels that emerge in protozoa operate in the human nervous system, whereas a variety of cell adhesion molecules are newly “invented” during evolution, enabled by an increase in gene numbers, alternative splice forms and transcription factors. Thereby, important regulatory and signalling molecules are retained as a protozoan heritage. published

Molecular cascades responsible for Ca(2+) homeostasis and Ca(2+) signalling could be assembled in highly plastic toolkits that define physiological adaptation of cells to the environment and which are intimately involved in all types of cellular pathology. Control over Ca(2+) concentration in different cellular compartments is intimately linked to cell metabolism, because (i) ATP production requires low Ca(2+), (ii) Ca(2+) homeostatic systems consume ATP and (iii) Ca(2+) signals in mitochondria stimulate ATP synthesis being an essential part of excitation-metabolic coupling. The communication between the ER and mitochondria plays an important role in this metabolic fine tuning. In the insulin resistance state and diabetes this communication has been impaired leading to different disorders, for instance, diminished insulin production by pancreatic β cells, reduced heart and skeletal muscle contractility, reduced NO production by endothelial cells, increased glucose production by liver, increased lipolysis by adipose cells, reduced immune responses, reduced cognitive functions, among others. All these processes eventually trigger degenerative events resulting in overt diabetes due to reduction of pancreatic β cell mass, and different complications of diabetes, such as retinopathy, nephropathy, neuropathy, and different cardiovascular diseases.

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca(2+) deregulation. Recently, we proposed a mechanism by which nanomolar Aβ42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca(2+) signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aβ42 on Ca(2+) signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1β, LPS). Here we report that Aβ42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca(2+) signals and store operated Ca(2+) entry (SOCE) were augmented in Aβ42-treated cells due to up-regulation of a set of Ca(2+) signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1β and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aβ42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aβ42 on astrocytic Ca(2+) signalling differ from and may contrast to the effects of pro-inflammatory agents.

Canonical transient receptor potential 1 (TRPC1) plasmalemmal cation channels mediate Ca2+ and Na+ fluxes and control respective cytoplasmic ion signals in rat cortical astrocytes. Mechanical stimulation of astrocytes results in an increase in the levels of cytosolic Ca2+ and Na+ that are in part due to entry of extracellular cations through TRPC1 containing channels. Inhibition of the TRPC1 pore with an antibody against its selective filter reduced cytosolic Ca2+ accumulation caused by mechanical stimulation. In contrast, this immunological treatment increased the cytosolic Na+ peak accumulation induced by mechanical stimulation. We propose that TRPC channels are amenable to changes in selective filtering, as mutations in previous studies and antibody binding in our present study differentially affect the flux of Ca2) and Na+. TRPC1 containing channels might represent focal points for co-ordination of Ca2+ and Na+ signalling in astroglia and this can have consequences on Ca(2+)- and Na(+)-dependent processes such as regulated exocytosis and lactate production, respectively, which in turn can modulate neuronal synaptic transmission.

Simultaneous recording of cytosolic and sarco-endoplasmic reticulum (SR/ER) luminal free calcium concentrations ([Ca(2+)](i) and [Ca(2+)](L), respectively) supports the notion that release channels (RyRs and IP(3)Rs) use a concealed Ca(2+) source, likely to be associated with intra-SR/ER Ca(2+) binding proteins, whereas SR/ER Ca(2+) leak channels can only access free luminal Ca(2+). We hypothesize that Ca(2+) is trapped by oligomers of luminal Ca(2+)-binding proteins and that the opening of release channels induces the rapid liberation of this "concealed" Ca(2+) source associated with intra-ER Ca(2+) buffers. Our hypothesis may also clarify why SERCA pumps potentiate Ca(2+) release and explain quantal characteristics and refractory states of Ca(2+) release process.

The evolution of the central nervous system (CNS) resulted in an appearance of highly specialised neuronal networks optimised for rapid information transfer. In the course of this specialisation neuronal cells lost their metabolic independence and the ability to survive in the absence of homeostatic systems. These homeostatic systems, represented by neuroglia regulate all aspects of CNS function in physiological and pathological conditions. The neurological diseases should be therefore considered as primary gliopathologies, which determine the progression and outcome of neuropathological process. Glial function is intimately regulated by cellular calcium signalling that underlies the specific form of "glial calcium excitability". Glial Ca2+ signals are triggered by activation of multiple receptors, and are primarily driven by Ca2+ release from the endoplasmic reticulum. In this review we summarise the role of glial calcium signalling in various forms of pathological processes including neurological and psychiatric disorders and neurodegeneration.

One of the most crucial aspects of Ca(2+) signalling is the ability to generate highly localised transient elevations of the cytosolic Ca(2+) concentration at specific strategically important target sites. Inevitably this necessitates a relatively high Ca(2+) buffering power of the cytoplasm, which in turn makes movement of Ca(2+) from one part of a cell to another difficult. Nature has evolved an elegant solution to this problem by creating operational Ca(2+) tunnels through the endoplasmic reticulum. Very recently direct evidence that such tunnelling also occurs in neurons has been provided.

Specific Ca(2+) homeostatic system appeared very early in the history of the cell, as a survival system preventing Ca(2+)-mediated cell damage. This homeostatic system produced a steep ( approximately 20,000 times) concentration gradient between extracellular and intracellular compartments, which has both survival importance (even relatively short increases in cytosolic Ca(2+) concentrations higher then 100 nM are incompatible with life) and signalling function. Evolution utilised this gradient together with an ability of Ca(2+) to interact with many biological molecules to create the most widespread and versatile signalling system, controlling the majority of cellular processes and executing complex routines of intercellular communications.

T-type, or low-voltage-activated (LVA), tiny and transient Ca2+ currents pare more and more recognised as universal players in a plethora of cell functions and are also more and more connected to several diseases. This short introduction reviews the discovery of T-type Ca2+ channels, describes its basic properties and sketches its cloning and physiological impact. Finally, an overview is given how research on T-type Ca2+ channels has developed in the last years and in which topics it is branching out, a process, which is still ongoing.

The nature of brain ageing and the age-dependent decline in cognitive functions remains poorly understood. Physiological brain ageing is characterised by mild mental dysfunctions, whereas age-dependent neurodegeneration, as illustrated by Alzheimer disease (AD), results rapidly in severe dementia. These two states of the aged brain, the physiological and the pathological, are fundamentally different as the latter stems from significant neuronal loss, whereas the former develops without significant neuronal demise. In this paper, we review the changes in neuronal Ca(2+) homeostasis that occur during brain ageing, and conclude that normal, physiological ageing is characterised mainly by a decrease of neuronal homeostatic reserve, defined as the capacity to respond effectively to functional and metabolic stressors, but does not reach the trigger required to induce neuronal death. In contrast, during neurodegenerative states, Ca(2+) homeostasis is affected early during the pathological process and result in significant neuronal demise. We also review recent evidence suggesting that the endoplasmic reticulum (ER) might play an important role in controlling the balance between healthy and pathological neuronal ageing.

The endoplasmic reticulum (ER) is an extensive organelle network formed by cisternae and microtubules, which stretches from the nuclear envelope to the cell surface in all eukaryotic cells. For many years, the ER was accepted as an intracellular “skeleton” and the place of protein synthesis. Recent discoveries have contributed to a growing appreciation for the role of the ER in co-ordinating extracellular and intracellular signals with long-term adaptive responses. Firstly, the ER was identified as a source for fast physiological signalling as a dynamic Ca2+ reservoir, which can be activated as a consequence of both electrical and chemical and cell stimulation. As such, the ER plays a crucial role in the propagation of cytosolic Ca2+ waves and and it can also function as an intracellular Ca2+ tunnel, allowing movement of ions through its lumen. Secondly, the classical work of Palade demonstrated that the ER serves as a common transport route by which secretory proteins are delivered to their destination. Further developments in this area have shown that the ER acts as a specialised environment for post-translational processing and targeting of many other types of proteins. Numerous chaperones resident within the ER lumen form various conformational intermediates, which determine the trafficking of proteins to the place of their final abode. Finally, it has become clear, that not only does the ER provide a place for protein folding and transport, but it also actively co-ordinates this process with the cellular status and physiological activity. This co-ordination is achieved by several signalling pathways originating from within the ER, thus coupling processes in the ER with those in the nucleus, which regulate gene expression. Consequently, the ER network can be regarded as a multi-functional organelle able to sense, convey, integrate and generate signalling events that are profoundly important for cellular physiology.

Peripheral sensory nervous system is comprised of neurones with their axons and neuroglia that includes satellite glial cells in sensory ganglia, myelinating, non-myelinating and perisynaptic Schwann cells. Pathogenesis of peripheral diabetic polyneuropathies is associated with aberrant function of both neurones and glia. Deregulated Ca(2+) homoeostasis and aberrant Ca(2+) signalling in neuronal and glial elements contributes to many forms of neuropathology and is fundamental to neurodegenerative diseases. In diabetes both neurones and glia experience metabolic stress and mitochondrial dysfunction which lead to deregulation of Ca(2+) homeostasis and Ca(2+) signalling, which in their turn lead to pathological cellular reactions contributing to development of diabetic neuropathies. Molecular cascades responsible for Ca(2+) homeostasis and signalling, therefore, can be regarded as potential therapeutic targets.