Neuronal Stores Research Articles

STIM Proteins: The Gas and Brake of Calcium Entry in Neurons.

Stromal interaction molecules (STIM)s are Ca2+ sensors in internal Ca2+ stores of the endoplasmic reticulum. They activate the store-operated Ca2+ channels, which are the main source of Ca2+ entry in non-excitable cells. Moreover, STIM proteins interact with other Ca2+ channel subunits and active transporters, making STIMs an important intermediate molecule in orchestrating a wide variety of Ca2+ influxes into excitable cells. Nevertheless, little is known about the role of STIM proteins in brain functioning. Being involved in many signaling pathways, STIMs replenish internal Ca2+ stores in neurons and mediate synaptic transmission and neuronal excitability. Ca2+ dyshomeostasis is a signature of many pathological conditions of the brain, including neurodegenerative diseases, injuries, stroke, and epilepsy. STIMs play a role in these disturbances not only by supporting abnormal store-operated Ca2+ entry but also by regulating Ca2+ influx through other channels. Here, we review the present knowledge of STIMs in neurons and their involvement in brain pathology.

Differential stability of task variable representations in retrosplenial cortex

Cortical neurons store information across different timescales, from seconds to years. Although information stability is variable across regions, it can vary within a region as well. Association areas are known to multiplex behaviorally relevant variables, but the stability of their representations is not well understood. Here, we longitudinally recorded the activity of neuronal populations in the mouse retrosplenial cortex (RSC) during the performance of a context-choice association task. We found that the activity of neurons exhibits different levels of stability across days. Using linear classifiers, we quantified the stability of three task-relevant variables. We find that RSC representations of context and trial outcome display higher stability than motor choice, both at the single cell and population levels. Together, our findings show an important characteristic of association areas, where diverse streams of information are stored with varying levels of stability, which may balance representational reliability and flexibility according to behavioral demands.

Deep convolutional neural network and IoT technology for healthcare.

Deep convolutional neural network and IoT technology for healthcare.

Energy optimisation predicts the capacity of ion buffering in the brain.

Neurons store energy in the ionic concentration gradients they build across their cell membrane. The amount of energy stored, and hence the work the ions can do by mixing, can be enhanced by the presence of ion buffers in extra- and intracellular space. Buffers act as sources and sinks of ions, however, and unless the buffering capacities for different ion species obey certain relationships, a complete mixing of the ions may be impeded by the physical conditions of charge neutrality and isotonicity. From these conditions, buffering capacities were calculated that enabled each ion species to mix completely. In all valid buffer distributions, the [Formula: see text] ions were buffered most, with a capacity exceeding that of [Formula: see text] and [Formula: see text] buffering by at least an order of magnitude. The similar magnitude of the (oppositely directed) [Formula: see text] and [Formula: see text] gradients made extracellular space behave as a [Formula: see text]-[Formula: see text] exchanger. Anions such as [Formula: see text] were buffered least. The great capacity of the extra- and intracellular [Formula: see text] buffers caused a large influx of [Formula: see text] ions as is typically observed during energy deprivation. These results explain many characteristics of the physiological buffer distributions but raise the question how the brain controls the capacity of its ion buffers. It is suggested that neurons and glial cells, by their great sensitivity to gradients of charge and osmolarity, respectively, sense deviations from electro-neutral and isotonic mixing, and use these signals to tune the chemical composition, and buffering capacity, of the extra- and intracellular matrices.

STAT3 transcriptionally regulates the expression of genes related to glycogen metabolism in developing motor neurons.

Motor neurons in the spinal cord are essential for movement. During the embryonic period, developing motor neurons store glycogen to protect against hypoglycemic and hypoxic stress. However, the mechanisms by which glycogen metabolism is regulated in motor neurons remain unclear. We herein investigated the transcriptional regulation of genes related to glycogen metabolism in the developing spinal cord. We focused on the regulatory mechanism of glycogen synthase (Gys1) and glycogen phosphorylase brain isoform (PygB), which play central roles in glycogen metabolism, and found that the transcription factor STAT3 regulated the expression of Gys1 and PygB via cis-regulatory promoter sequences in the developing spinal cord. These results suggest that STAT3 is important for the regulation of glycogen metabolism during motor neuron development.

Neurophysiological functions and pharmacological tools of acidic and non-acidic Ca2+ stores

Ca2+ signalling is of prime importance in controlling numerous cell functions in the brain. Endolysosomes are acidic organelles currently emerging as important Ca2+ stores in astrocytes, microglia, endothelial cells, and neurons. In neurons, these acidic Ca2+ stores are found in axons, soma, dendrites, and axon endings and could provide local sources of Ca2+ to control synaptic transmission, neuronal plasticity, and autophagy to name a few. This review will address how acidic Ca2+ stores are recruited in response to cell stimulation. We will focus on the role of the endolysosomal two-pore channels (TPCs) and their physiological agonist nicotinic acid adenine dinucleotide phosphate (NAADP) and how they interact with cyclic ADP-ribose and ryanodine receptors from the endoplasmic reticulum. Finally, this review will describe new pharmacological tools and animal mutant models now available to explore acidic Ca2+ stores as key elements in brain function and dysfunction.

Exploring the Pivotal Neurophysiologic and Therapeutic Potentials of Vitamin C in Glioma

Gliomas represent solely primary brain cancers of glial cell or neuroepithelial origin. Gliomas are still the most lethal human cancers despite modern innovations in both diagnostic techniques as well as therapeutic regimes. Gliomas have the lowest overall survival rate compared to other cancers 5 years after definitive diagnosis. The dietary intake of vitamin C has protective effect on glioma risk. Vitamin C is an essential compound that plays a vital role in the regulation of lysyl and prolyl hydroxylase activity. Neurons store high levels of vitamin C via sodium dependent-vitamin C transporters (SVCTs) to protect them from oxidative ischemia-reperfusion injury. Vitamin C is a water-soluble enzyme, typically seen as a powerful antioxidant in plants as well as animals. The key function of vitamin C is the inhibition of redox imbalance from reactive oxygen species produced via the stimulation of glutamate receptors. Gliomas absorb vitamin C primarily via its oxidized dehydroascorbate form by means of GLUT 1, 3, and 4 and its reduced form, ascorbate, by SVCT2. Vitamin C is able to preserve prosthetic metal ions like Fe2+ and Cu+ in their reduced forms in several enzymatic reactions as well as scavenge free radicals in order to safeguard tissues from oxidative damage. Therapeutic concentrations of vitamin C are able to trigger H2O2 generation in glioma. High-dose combination of vitamin C and radiation has a much more profound cytotoxic effect on primary glioblastoma multiforme cells compared to normal astrocytes. Control trials are needed to validate the use of vitamin C and standardization of the doses of vitamin C in the treatment of patients with glioma.

Selective linkage of mitochondrial enzymes to intracellular calcium stores differs between human-induced pluripotent stem cells, neural stem cells, and neurons.

Mitochondria and releasable endoplasmic reticulum (ER) calcium modulate neuronal calcium signaling, and both change in Alzheimer's disease (AD). The releasable calcium stores in the ER are exaggerated in fibroblasts from AD patients and in multiple models of AD. The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme complex, is diminished in brains from AD patients, and can be plausibly linked to plaques and tangles. Our previous studies in cell lines and mouse neurons demonstrate that reductions in KGDHC increase the ER releasable calcium stores. The goal of these studies was to test whether the relationship was true in human iPSC-derived neurons. Inhibition of KGDHC for one or 24hr increased the ER releasable calcium store in human neurons by 69% and 144%, respectively. The effect was mitochondrial enzyme specific because inhibiting the pyruvate dehydrogenase complex, another key mitochondrial enzyme complex, diminished the ER releasable calcium stores. The link of KGDHC to ER releasable calcium stores was cell type specific as the interaction was not present in iPSC or neural stem cells. Thus, these studies in human neurons verify a link between KGDHC and releasable ER calcium stores, and support the use of human neurons to examine mechanisms and potential therapies for AD.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.

Calsequestrins New Calcium Store Markers of Adult Zebrafish Cerebellum and Optic Tectum.

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.

SK2 channels in cerebellar Purkinje cells contribute to excitability modulation in motor-learning-specific memory traces.

Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such “intrinsic plasticity” in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell–specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning.

Effects of enriched housing on the neuronal morphology of mice that lack zinc transporter 3 (ZnT3) and vesicular zinc

In the central nervous system, certain neurons store zinc within the synaptic vesicles of their axon terminals. This vesicular zinc can then be released in an activity-dependent fashion as an intercellular signal. The functions of vesicular zinc are not entirely understood, but evidence suggests that it is important for some forms of experience-dependent plasticity in the brain. The ability of neurons to store and release vesicular zinc is dependent on expression of the vesicular zinc transporter, ZnT3. Here, we examined the neuronal morphology of mice that lack ZnT3. Brains were collected from mice housed under standard laboratory conditions and from mice housed in enriched environments – large, multilevel enclosures with running wheels, numerous objects and tunnels, and a greater number of cage mates. Golgi-Cox staining was used to visualize neurons for analysis of dendritic length and dendritic spine density. Neurons were analyzed from the barrel cortex, striatum, basolateral amygdala, and hippocampus (CA1). ZnT3 knockout mice, relative to wild type mice, exhibited increased basal dendritic length in the layer 2/3 pyramidal neurons of barrel cortex, independently of housing condition. Environmental enrichment decreased apical dendritic length in these same neurons and increased dendritic spine density on striatal medium spiny neurons. Elimination of ZnT3 did not modulate any of the effects of enrichment. Our results provide no evidence that vesicular zinc is required for the experience-dependent changes that occur in response to environmental enrichment. They are consistent, however, with recent reports suggesting increased cortical volume in ZnT3 knockout mice.

A Feedforward Mechanism for Epileptogenesis Regulated by Lactate Dehydrogenase A

Abstract INTRODUCTION Despite the ketogenic diet's successful use since the 1920s, epilepsy as a disease of energy metabolism is a novel concept. We previously established that seizures deplete neuronal energy stores and reprogram neurons from aerobic to glycolytic metabolic phenotype, marked by upregulation of lactate dehydrogenase A (LDHA). LDHA has recently been shown to play a role in neuronal membrane depolarization and epileptogenesis. We show here that LDHA upregulation through HIF1a may lead to seizure formation. METHODS Resected tissue from 11 epileptic patients were probed for LDHA expression. To study the electrophysiological consequences of LDHA, we used a mixed rat cortical cell culture model on a microelectrode array (MEA). Furthermore, we developed a novel murine model of chronic focal cortical epilepsy to establish HIF1a's role in mediating LDHA expression and seizure formation. Finally, we used a lentivirus vector to directly upregulate LDHA in neurons cultured on an MEA to measure neuronal bursting. RESULTS We found LDHA increased significantly in epileptic tissue versus nonepileptic tissue. Induction of seizure activity in cultured neurons with low magnesium resulted in increased LDHA and subsequently increased baseline bursting over 10 d. Inhibition of LDHA activity with stiripentol and isosafrole decreased the overall burst frequency. Cells that were induced to upregulate LDHA via DMOG, an upstream HIF1a potentiator, showed a significant increase in baseline bursting activity. Furthermore, placement of cobalt, a HIF1a stabilizer, into the frontal cortex of mice caused chronic seizures emanating from perilesion cortex which showed increased LDHA. Finally, direct LDHA upregulation resulted in increased bursting activity confirming that LDHA may lead to seizure formation. CONCLUSION Overall, our data show that LDHA, regulated by HIF1a, can contribute to seizure development. These data suggest a novel molecular mechanism for the pathogenesis of epilepsy where seizures cause LDHA upregulation which then further drives seizures, leading to a cycle of epileptogenesis.

Two types of functionally distinct Ca2+ stores in hippocampal neurons

It is widely assumed that inositol trisphosphate (IP3) and ryanodine (Ry) receptors share the same Ca2+ pool in central mammalian neurons. We now demonstrate that in hippocampal CA1 pyramidal neurons IP3- and Ry-receptors are associated with two functionally distinct intracellular Ca2+ stores, respectively. While the IP3-sensitive Ca2+ store refilling requires Orai2 channels, Ry-sensitive Ca2+ store refilling involves voltage-gated Ca2+ channels (VGCCs). Our findings have direct implications for the understanding of function and plasticity in these central mammalian neurons.

Sigma 1 receptor activation modifies intracellular calcium exchange in the G93AhSOD1 ALS model

Aberrations in intracellular calcium (Ca2+) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca2+ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca2+ cycle (ERMCC). The ER supplies Ca2+ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca2+ at the cellular level remains to be thoroughly investigated.Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca2+ homeostasis in the presence of G93AhSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca2+ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca2+ clearance following a) AMPAR activation by kainate and b) IP3R–mediated ER Ca2+ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca2+. Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca2+ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca2+ homeostasis.

Activity-Dependent Exocytosis of Lysosomes Regulates the Structural Plasticity of Dendritic Spines

SummaryLysosomes have traditionally been viewed as degradative organelles, although a growing body of evidence suggests that they can function as Ca2+ stores. Here we examined the function of these stores in hippocampal pyramidal neurons. We found that back-propagating action potentials (bpAPs) could elicit Ca2+ release from lysosomes in the dendrites. This Ca2+ release triggered the fusion of lysosomes with the plasma membrane, resulting in the release of Cathepsin B. Cathepsin B increased the activity of matrix metalloproteinase 9 (MMP-9), an enzyme involved in extracellular matrix (ECM) remodelling and synaptic plasticity. Inhibition of either lysosomal Ca2+ signaling or Cathepsin B release prevented the maintenance of dendritic spine growth induced by Hebbian activity. This impairment could be rescued by exogenous application of active MMP-9. Our findings suggest that activity-dependent exocytosis of Cathepsin B from lysosomes regulates the long-term structural plasticity of dendritic spines by triggering MMP-9 activation and ECM remodelling.

Ventral CA1 neurons store social memory.

The medial temporal lobe, including the hippocampus, has been implicated in social memory. However, it remains unknown which parts of these brain regions and their circuits hold social memory. Here, we show that ventral hippocampal CA1 (vCA1) neurons of a mouse and their projections to nucleus accumbens (NAc) shell play a necessary and sufficient role in social memory. Both the proportion of activated vCA1 cells and the strength and stability of the responding cells are greater in response to a familiar mouse than to a previously unencountered mouse. Optogenetic reactivation of vCA1 neurons that respond to the familiar mouse enabled memory retrieval and the association of these neurons with unconditioned stimuli. Thus, vCA1 neurons and their NAc shell projections are a component of the storage site of social memory.

Recoding a cocaine-place memory engram to a neutral engram in the hippocampus.

The hippocampus provides the brain’s memory system with a subset of neurons holding a map-like representation of each environment experienced. We found in mice that optogenetic-silencing those neurons active in an environment unmasked a subset of quiet neurons, enabling the emergence of an alternative map. This intervention applied in a cocaine-paired environment neutralized an otherwise long-lasting drug-place preference, showing that recoding a spatial memory engram can alleviate associated maladaptive behavior.

Formalin evokes calcium transients from the endoplasmatic reticulum.

The formalin test is the most widely used behavioral screening test for analgesic compounds. The cellular mechanism of action of formaldehyde, inducing a typically biphasic pain-related behavior in rodents is addressed in this study. The chemoreceptor channel TRPA1 was suggested as primary transducer, but the high concentrations used in the formalin test elicit a similar response in TRPA1 wildtype and knockout animals. Here we show that formaldehyde evokes a dose-dependent calcium release from intracellular stores in mouse sensory neurons and primary keratinocytes as well as in non-neuronal cell lines, and independent of TRPA1. The source of calcium is the endoplasmatic reticulum and inhibition of the sarco/endoplasmic reticulum calcium-ATPase has a major contribution. This TRPA1-independent mechanism may underlie formaldehyde-induced pan-neuronal excitation and subsequent inflammation.

Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons

Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50Hz, 1mT or 3mT, lasting 24h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K+-evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca2+ ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channel-independent mechanism.