Calcium Concentration In Neurons Research Articles

Age-related deficits in neuronal physiology and cognitive function are recapitulated in young mice overexpressing the L-type calcium channel, CaV 1.3.

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, CaV 1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of CaV 1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of CaV 1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.

A multiple timescale network model of intracellular calcium concentrations in coupled neurons: Insights from ROM simulations

In Fernández-García and Vidal [Physica D 401 (2020) 132129], the authors have analyzed the synchronization features between two identical 3D slow-fast oscillators, symmetrically coupled, and built as an extension of the FitzHugh—Nagumo dynamics generating Mixed-Mode Oscillations. The third variable in each oscillator aims at representing the time-varying intracellular calcium concentration in neurons. The global model is therefore six-dimensional with two fast variables and four slow variables with strong symmetry properties. In the present article, we consider an extension of this model in two different directions. First, we consider heterogeneity among cells and analyze the coupling of two oscillators with different values for one parameter which tunes the intrinsic frequency of the output. We therefore identify new patterns of antiphasic synchronization, with non trivial signatures and that exhibit a Devil’s Staircase phenomenon in signature transitions when varying the coupling gain parameter value. Second, we introduce a network of N cells divided into two clusters: the coupling between neurons in each cluster is excitatory, while the coupling between the two clusters is inhibitory. Such system aims at modelling the interactions between neurons tending to synchronization in each of two subpopulations that inhibit each other, like ipsi- and contra-lateral motoneurons assemblies. To perform the numerical simulations in this case when N is large, as an initial step towards the network analysis, we consider Reduced Order Models in order to save computational costs. We present the numerical reduction results in a network of 100 cells. For the sake of validation of the numerical reduction method, we both compare the outputs and CPU times obtained with the original and the reduced models in different cases of network coupling structures.

Mathematical Modeling of Calcium Oscillatory Patterns in a Neuron.

Calcium oscillations are an imperative mode of signaling phenomenon. These oscillations are due to the active interactions taking place between some of the parameters like voltage gated calcium channels (VGCC), sodium calcium exchanger (NCX), calcium binding buffers, endoplasmic reticulum (ER) and mitochondria. The present paper focuses on the problem of higher level of calcium concentration in neurons which may further result into Alzheimer's Disease (AD). For this, a three-dimensional mathematical model having a system of differential equations depicting the changes in cytosolic calcium (in presence of buffers, VGCC and NCX), ER calcium and mitochondrial calcium, is formulated. A three-dimensional neuronal structure is targeted as the domain which is further discussed and solved using finite element technique in Comsol Multiphysics 5.4. Apposite boundary conditions matching well with the in-situ conditions are assumed. The obtained results clearly show the significance of the lower amount of the buffer and higher calcium mediated activities of VGCC, NCX, ER and mitochondria on calcium profile. These changes may lead to AD. To transit from AD condition to normal, exogenous buffers are added to check their significance. The results thus show that the replenishment of buffer may balance the amount of cell calcium and hence can affect positively on Alzheimer's affected cells.

Decreased expression of lncRNA Malat1 in rat spinal cord contributes to neuropathic pain by increasing neuron excitability after brachial plexus avulsion.

Purpose: Neuropathic pain (NP) is a challenging clinical problem due to its complex pathogenesis. In our previous study using microarray, we found that the levels of lncRNA Malat1 were decreased in the spinal cord of NP rat after brachial plexus avulsion, but its contribution to NP remain unclear. The purpose of this study was to investigate its role in the pathogenesis of NP.Methods: In the NP model of complete brachial plexus avulsion rat, spinal cords were harvested, and fluorescence in situ hybridization (FISH) was used to test the spatial expression of Malat1 and qRT-PCR was used to confirm the quantitative expression of Malat1. In primary cultured neurons, Malat1 expression interfered with adenovirus. Spontaneous electric activities of neurons were tested using multi-electrode arrays and apoptosis of neurons was tested using TUNEL method. The change of intracellular calcium concentration was analyzed using calcium imaging method.Results: Decreased Malat1 expression was confirmed using qRT-PCR, and Malat1 was identified in the cytoplasm of neurons in spinal cord, but not in glia. In vitro, the decrease of Malat1 resulted in an increase in the frequency of spontaneous electric activity in neurons but had no effect on neuronal apoptosis. Further analysis indicated during glutamate stimulation, the change of intracellular calcium concentration in neurons with downregulated Malat1 expression was significantly greater than that in normal neurons.Conclusion: Reduced Malat1 expression may induce NP by increasing neuronal excitability in the spinal cord via regulation of calcium flux.

Effects of drinking water fluorosis on L-type calcium channel of hippocampal neurons in mice

The study aimed to investigate the effects of drinking water fluorosis on L-type calcium channels (LTCCs) in mouse hippocampal neurons. A total of 60 newly weaned ICR male mice were randomly divided into control, low fluoride and high fluoride groups. After 3 and 6 months of exposure to fluoride, the patch clamp technique was used to detect the peak and relative values (I/Imax), steady-state activation curve ratio (G/Gmax), decay time constant, and tail current time constant of LTCCs currents in hippocampal CA1 region of mouse brain slices. Fluoride greatly reduced the serum and urinary calcium concentrations in mice, and the chronic fluorosis has a greater impact than subchronic fluorosis. The peak value of LTCCs current in pyramidal neurons of hippocampal CA1 area was significant and increased with the prolonged exposure time. The relative values of current and steady-state coefficients were changed greatly. The decay and tail current time increased significantly. High fluorine concentration indicates great peak value and open time of LTCCs opening. LTCCs are sensitive to fluoride exposure. The activation voltage of calcium channels induced by fluoride exposure is decreased, the opening time of calcium channels is prolonged, and the calcium influx per unit time increased, thereby overloading calcium concentration in neurons and this may be an explanation for intracellular calcium overload caused by fluoride. The imbalance of calcium metabolism caused by fluorosis may be a pathogenesis of brain injury induced by fluoride. Furthermore, the risk of brain damage from low-fluorine exposure cannot be ignored.

Imidazole improves cognition and balances Alzheimer's-like intracellular calcium homeostasis in transgenic Drosophila model.

The characteristic of Alzheimer's disease (AD) is the accumulation and aggregation of amyloid-β (Aβ). So far, we already know that the dysregulation of intracellular calcium homeostasis is considered to be associated with Aβ neurotoxicity. Meantime, we also found that the channels formed by Aβ are electronegative as calcium channels. Base on this hypothesis, the formation of Aβ channels will provide us with a new therapeutic direction for AD. Aβ channel hypothesis is proposed that the axis of Aβ channel's pore was encompasses by the His13 -His14 diad. Imidazole especially the imidazole ring was supposed binding to the side chains of Aβ peptides. In our study, we adopted Gal4/UAS system to establish transgenic drosophila model which lay a good foundation to explore the imidazole's function and mechanism of action. The results suggested that Imidazole could not only improve the cognition of Aβ42-expressing flies, but also decreases p-JNK activation in whole brain of Aβ42-expressing flies. Furthermore, freshly prepared oligomeric Aβ42 peptide ascended primary pupal neuronal calcium concentration and this phenomenon was alleviated by Imidazole and Zn2+ .

The relevance of kalikrein-kinin system via activation of B2 receptor in LPS-induced fever in rats

PurposeThis study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B1 and B2 receptors on LPS- induced fever and the POA cells involved in this response. Material and methodsMale Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca++ signaling in POA cells was performed in rat pup brain tissue microcultures. ResultsIcatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE2concentration. Effect abolished by indomethacin (i.p.). ConclusionsLPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B2-receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B2-receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever.

Low-Dose Lithium Stabilizes Human Endothelial Barrier by Decreasing MLC Phosphorylation and Universally Augments Cholinergic Vasorelaxation Capacity in a Direct Manner.

Lithium at serum concentrations up to 1 mmol/L has been used in patients suffering from bipolar disorder for decades and has recently been shown to reduce the risk for ischemic stroke in these patients. The risk for stroke and thromboembolism depend not only on cerebral but also on general endothelial function and health; the entire endothelium as an organ is therefore pathophysiologically relevant. Regardless, the knowledge about the direct impact of lithium on endothelial function remains poor. We conducted an experimental study using lithium as pharmacologic pretreatment for murine, porcine and human vascular endothelium. We predominantly investigated endothelial vasorelaxation capacities in addition to human basal and dynamic (thrombin-/PAR-1 receptor agonist-impaired) barrier functioning including myosin light chain (MLC) phosphorylation (MLC-P). Low-dose therapeutic lithium concentrations (0.4 mmol/L) significantly augment the cholinergic endothelium-dependent vasorelaxation capacities of cerebral and thoracic arteries, independently of central and autonomic nerve system influences. Similar concentrations of lithium (0.2–0.4 mmol/L) significantly stabilized the dynamic thrombin-induced and PAR-1 receptor agonist-induced permeability of human endothelium, while even the basal permeability appeared to be stabilized. The lithium-attenuated dynamic permeability was mediated by a reduced endothelial MLC-P known to be followed by a lessening of endothelial cell contraction and paracellular gap formation. The well-known lithium-associated inhibition of inositol monophosphatase/glycogen synthase kinase-3-β signaling-pathways involving intracellular calcium concentrations in neurons seems to similarly occur in endothelial cells, too, but with different down-stream effects such as MLC-P reduction. This is the first study discovering low-dose lithium as a drug directly stabilizing human endothelium and ubiquitously augmenting cholinergic endothelium-mediated vasorelaxation. Our findings have translational and potentially clinical impact on cardiovascular and cerebrovascular disease associated with inflammation explaining why lithium can reduce, e.g., the risk for stroke. However, further clinical studies are warranted.

Protective effects of GTM-1 on endoplasmic reticulum stress induced by thapsgargin in rat neurons

GTM-1 is a drug that reverses Alzheimer's Disease (AD) development specifically induced by thapsgargin (TG) and endoplasmic reticulum (ER) has been reported to be a pilot process that leads to AD formation. It is speculated that GTM-1 could also prohibit TG-induced ER stress. In this study, we utilized immuno-fluorescence to identify morphological changes in nucleus and transmission electron microscopy was used to observe neuronal ultra-structures. Moreover, expressions of GRP78, CHOP, Bcl-2 and cytochrome c were assessed using immuno-blotting, while calcium concentration was detected by fluorescence spectrometer. As suggested by the above cellular experiments, neuronal ultrastructures were damaged by the treatment of TG, while this damaging trend was reversed when neurons were simultaneously treated with both TG and GTM-1. Besides that, certain marker proteins of ER stress (e.g. GRP78, CHOP, and cytochrome c) and calcium concentrations in neurons were significantly increased when TG was applied, while these levels were reduced to normal conditions when GTM-1 was added in the treatment. In conclusion, GTM-1 restrained the ongoing of ER stress that was induced by TG.

Suppression of STIM1 in the early stage after global ischemia attenuates the injury of delayed neuronal death by inhibiting store-operated calcium entry-induced apoptosis in rats

Ca²⁺ overload is considered to be the most important ion imbalance in the neuronal injury. Store-operated Ca²⁺ entry has been suggested to be a significant mechanism of excessive Ca²⁺ influx in many cells. The role of store-operated Ca²⁺ entry in neuronal ischemic injury has yet to be elucidated. The aim of this study was to assess the role of store-operated calcium channel (SOCC) proteins involved with calcium overload in the induction of delayed neuronal death after global ischemia in rats. A transient RNA interference model of global ischemia in rats was established to determine the role of SOCC-induced Ca²⁺ overload in delayed neuronal death. We found that STIM1 and ORAI1 expression in the hippocampus increased continuously after global ischemia and peaked on day 4. These data were consistent with an increase in the intracellular calcium concentration. Using Stim1 siRNA to suppress SOCC activity in the early stage of ischemia significantly inhibited STIM1 and ORAI1 expression and decreased the intracellular calcium concentration in neurons. In addition, the neurological function of rats improved after the Stim1 siRNA injection. High expression of STIM1 and ORAI may be the source of excessive calcium influx after ischemic damage. Blocking of this SOCC-induced calcium influx could lead to an improved neuronal survival. These data suggest that calcium influx through SOCC is another nonexcitotoxicity mechanism of ischemic neuronal death.

Nesfatin‐1 does not influence intracellular calcium concentrations in neurons of the nucleus of the solitary tract or the paraventricular nucleus

Nesfatin‐1 (nes‐1) is a neuropeptide which exerts potent effects on food intake, cardiovascular parameters, and the stress axis. Immunohistochemical studies have shown expression of nes‐1 in the nucleus of the solitary tract (NTS) and paraventricular nucleus (PVN). To date there is minimal data describing the mechanism of action of nes‐1 in the brain. Using whole cell patch clamp recordings, we have previously shown bath applied nes‐1(10 nM) influences the membrane potential of 63% of NTS and 71% of PVN neurons. Here, as a first step in investigating the mechanism of nes‐1 actions, we examined the effect of nes‐1 on intracellular Ca2+ concentrations ([Ca2+]i) in dissociated NTS and PVN neurons loaded with the Ca2+ indicator Fura2‐AM. Bath application of nes‐ 1 at 10 and 100 nM increased [Ca2+]i in 4% (n=75) and 3% (n=84) of NTS neurons, respectively, as assessed by a minimum 20% increase in the ratiometric value of [Ca2+]i. Furthermore, 9% (n=125) of PVN neurons showed an increase in [Ca2+]i in response to 10 nM nes‐1. Our findings show nes‐1 does not influence [Ca2+]i in NTS or PVN neurons. A large discrepancy also exists in the proportion of neurons reported as responsive to nes‐1 in our electrophysiological vs calcium imaging experiments, suggesting studies of nes‐1 effects on [Ca2+]i may not be an appropriate method of evaluating neuronal nes‐1 activation patterns or mechanisms of action.Funding: NSERC, FQRNT, HSFO.

Deactivation of L-type Ca Current by Inhibition Controls LTP at Excitatory Synapses in the Cerebellar Nuclei

Long-term potentiation (LTP) of mossy fiber EPSCs in the cerebellar nuclei is controlled by synaptic inhibition from Purkinje neurons. EPSCs are potentiated by a sequence of excitation, inhibition, and disinhibition, raising the question of how these stimuli interact to induce plasticity. Here, we find that synaptic excitation, inhibition, and disinhibition couple to different calcium-dependent signaling pathways. In LTP induction protocols, constitutively active calcineurin can replace synaptic excitation, and constitutively active alpha-CaMKII can replace calcium influx associated with resumption of spiking upon disinhibition. Additionally, nimodipine can replace hyperpolarization, indicating that inhibition of firing decreases Ca influx through L-type Ca channels, providing a necessary signal for LTP. Together, these data suggest that potentiation develops after a calcineurin priming signal combines with an alpha-CaMKII triggering signal if and only if L-type Ca current is reduced. Thus, hyperpolarization induced by synaptic inhibition actively controls excitatory synaptic plasticity in the cerebellar nuclei.

Slow Excitation of Cultured Rat Retinal Ganglion Cells by Activating Group I Metabotropic Glutamate Receptors

As in many CNS neurons, retinal ganglion cells (RGCs) receive fast synaptic activation through postsynaptic ionotropic receptors. However, the potential role of postsynaptic group I metabotropic glutamate receptors (mGluRs) in these neurons is unknown. In this study we first demonstrated that the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) increased intracellular calcium concentration in neurons within the ganglion cell layer of the rat retina. This prompted us to use an immunopanned-RGC and cortical astroglia coculture preparation to explore the effect of group I mGluR activation on the electrophysiological properties of cultured RGCs. Using perforated patch-clamp recordings in current-clamp configuration, we found that application of DHPG increased spontaneous spiking and depolarized the resting membrane potential of RGCs. This boosting effect was attributed to an increase in membrane resistance due to blockade of a background K(+) conductance. Further experiments showed that the group I mGluR-sensitive K(+) conductance was not blocked by 3 mM Cs(+), but was sensitive to acidification. Pharmacological studies indicated that the effect of DHPG on RGCs was mediated by the mGluR1 rather than the mGluR5 receptor subtype. Our results suggest a facilitatory role for group I mGluR activation in modulating RGC excitability in the mammalian inner retina.

Isoflurane Inhibits Growth but Does Not Cause Cell Death in Hippocampal Neural Precursor Cells Grown in Culture

Isoflurane causes long-term hippocampal-dependent learning deficits in rats despite limited isoflurane-induced hippocampal cell death, raising questions about the causality between isoflurane-induced cell death and isoflurane-induced cognitive function. Neurogenesis in the dentate gyrus is required for hippocampal-dependent learning and thus constitutes a potential alternative mechanism by which cognition can be altered after neonatal anesthesia. The authors tested the hypothesis that isoflurane alters proliferation and differentiation of hippocampal neural progenitor cells. Multipotent neural progenitor cells were isolated from pooled rat hippocampi (postnatal day 2) and grown in culture. These cells were exposed to isoflurane and evaluated for cell death using lactate dehydrogenase release, caspase activity, and immunocytochemistry for nuclear localization of cleaved caspase 3. Growth was assessed by cell counting and BrdU incorporation. Expression of markers of stemness (Sox2) and cell division (Ki67) were determined by quantitative polymerase chain reaction. Cell fate selection was assessed using immunocytochemistry to stain for neuronal and glial markers. Isoflurane did not change lactate dehydrogenase release, activity of caspase 3/7, or the amount of nuclear cleaved caspase 3. Isoflurane decreased caspase 9 activity, inhibited proliferation, and decreased the proportion of cells in s-phase. messenger ribonucleic acid expression of Sox2 (stem cells) and Ki67 (proliferation) were decreased. Differentiating neural progenitor cells more often select a neuronal fate after isoflurane exposure. The authors conclude that isoflurane does not cause cell death, but it does act directly on neural progenitor cells independently of effects on the surrounding brain to decrease proliferation and increase neuronal fate selection. These changes could adversely affect cognition after isoflurane anesthesia.

P3-405: Overexpression of human APP increases neuronal calcium influx through L-type calcium channels independently of Aβ or γ-cleavage

Calcium homeostasis is crucial for neuronal survival. A growing body of evidence shows that in Alzheimer disease, calcium homeostasis is disrupted, leading to abnormal synaptic function and memory deficits. Voltage-dependant calcium channels (VDCC) are influenced by Aβ and presenilins, but little is known about the contribution of the Amyloid Precursor Protein (APP) to calcium currents in neurons. Overexpression of human neuronal APP (hAPP) in primary cultures of rat cortical neurons was mediated by recombinant adenoviruses. Voltage-dependent calcium channels activity was recorded by the patch-clamp technique in the whole-cell configuration and cytosolic calcium concentration was measured using FURA-2. The density of L-type VDCC at the cell surface was measured in binding experiments using a radiolabelled ligand, [3H]PN200–110. BayK6844, a L-type calcium channel agonist, induced a larger increase in cytosolic calcium concentration in neurons expressing hAPP than in control (β-gal expressing) neurons. The increased response to BayK6844 was maintained after inhibition of Aβ production and γ-cleavage by DAPT, a functional inhibitor of the γ-secretase activity. The effect of APP could not be attributed to an increased number of L-type calcium channels at the cell surface, as measured by the binding of [3H]PN200–110 to hAPP or β-gal expressing neurons. Depolarization of neurons using 50mM KCl led to a similar increase in cytosolic Ca2+ concentration in hAPP or β-gal expressing neurons. Voltage-clamp recordings showed similar densities of calcium currents in hAPP expressing neurons, when compared to non-infected neurons. However, a shift of the voltage dependence of activation to more positive potentials was observed, which is compatible with a higher contribution of L-type calcium channels to the global calcium current. Neuronal expression of human APP increases the activity of L-type calcium channels, independently of Aβ production or γ-cleavage. This increase does not induce a higher total calcium current, contrary to previous observations, after treatment of neurons with synthetic Aβ. An important function of APP could be to regulate voltage-dependent calcium channels by favoring the L-type calcium channel contribution to total calcium currents.

Monitoring Calcium Concentration in Neurons with Cameleon

The calcium ion, a second messenger in the brain, plays key roles in neuronal signaling pathways. Ca(2+) signals in neurons are often highly localized and difficult to measure accurately. The quantification of calcium concentration is thus critical for understanding neuronal signaling. In this study, a yellow cameleon (YC3.60) excited using a 458 nm laser was used to monitor the calcium signals in neurons, and the dynamic range (R(max)/R(min)) of YC3.60 was found to reach 250%. The spatial distribution of calcium and the physiological changes in hippocampal neurons and even in spines were determined by the fluorescence resonance energy transfer (FRET) method. It was proved that cameleon could be used for the quantitative measurement of calcium concentration in neurons. Fluorescence readout of the calcium concentration in neurons by FRET is nondestructive, quantifiable with high spatiotemporal resolution, and even applicable to dendritic spines.

Converting Attraction to Repulsion

During nervous system development, axonal growth cones respond to attractive and repulsive cues that help them navigate to their targets. Wen et al . discovered that, whereas embryonic Xenopus spinal axons in an in vitro culture system were attracted to a gradient of bone morphogenetic protein 7 (BMP7) presented 4 to 8 hours after being placed in culture, BMP7 repelled the same axons after they had been cultured overnight. The type II BMP receptor (BMPRII) mediated both attractive and repulsive responses, and overexpression of a shortened form of BMPRII [which lacks a region that mediates interactions with LIM kinase I (LIMKI) but is dispensable for activation of Smad signaling] abolished BMP7-induced turning. A 32-amino-acid peptide that competitively inhibits LIMKI abolished attractive turning to BMP7, as did a dominant-negative LIMKI mutant, whereas neither affected repulsive turning. In neurons cultured overnight that overexpressed a dominant-negative mutant of the phosphatase Slingshot (SSH), however, the response to BMP7 was converted from repulsion to attraction. A combination of live imaging of the actin cytoskeleton, immunofluorescence analysis of the phosphorylation of XAC [ Xenopus actin-depolymerizing factor (ADF)/cofilin], and expression of wild-type and mutant forms of XAC revealed that LIMKI and SSH regulated growth cone turning through phosphorylation of XAC. BMP7 elicited an increase in growth cone calcium concentration in neurons cultured overnight but not in those cultured for 6 hours. Manipulations designed to block calcium signaling failed to affect attractive turning in 4- to 8-hour cultures, whereas in overnight cultures these treatments abolished the BMP-dependent decrease in XAC phosphorylation and converted repulsive to attractive turning, as did pharmacological inhibition of calcineurin. The transient receptor potential channel TRPC1 was more abundant in overnight cultures than in 4- to 8-hour cultures, and expression of a dominant-negative TRPC1 mutant abolished BMP7-dependent repulsion in overnight cultures, whereas overexpression of wild-type TRPC1 elicited BMP7-dependent repulsion in 4- to 8-hour cultures. Thus, opposing actions of LIMKI and SSH on XAC phosphorylation appear to regulate the actin cytoskeleton and thereby growth cone turning, with calcium influx through TRPC1 switching attraction to repulsion by shifting the balance toward SSH. Z. Wen, L. Han, J. R. Bamburg, S. Shim, G.-l. Ming, J. Q. Zheng, BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J. Cell Biol. 178 , 107-119 (2007). [Abstract] [Full Text]

Ratiometric calcium concentration estimation using LED excitation during mechanotransduction in single sensory neurons

In a previous study using Oregon Green BAPTA-1 fluorescence we found that intracellular calcium concentration in spider mechanoreceptor neurons rose during mechanical stimulation. We also showed that calcium elevation required the opening of voltage-dependent calcium channels by action potentials, and could not be produced by the receptor potential alone. While evidence for mechanisms of calcium elevation in these neurons was clear, our estimates of actual calcium concentration depended on properties of the fluorescent dye in the neuron cytoplasm that could not be verified. We have now developed a method for ratiometric estimation of calcium concentration in these neurons using Fura Red dye, excitation by two light emitting diodes (LEDs) of different wavelengths, and an avalanche photodiode fluorescence detector. The method is simple and economical to implement, allows concentration changes to be measured in the millisecond time range, and could easily be applied to a wide range of preparations. Resting calcium concentration in these neurons was about 70nM and rose to a maximum of about 400nM at firing rates above 20 action potentials per second.

T Cells Attacking Neurons

An autoimmune attack on oligodendrocytes and myelin has long been implicated in the pathogenesis of multiple sclerosis (MS) and related neuroinflammatory diseases. More recent data suggest that T cells that invade the brain during neuroinflammation target neurons as well. Moreover, neuronal damage correlates with the clinical course of MS. Using two-photon microscopy, Nitsch et al. investigated interactions between T cells and living neurons in freshly dissected mouse cortical slices. The authors found that fluorescently labeled T cells that had been stimulated with antigen (but not unstimulated T cells) rapidly entered the brain parenchyma and made contact with neurons. Contact with neuronal cell bodies or proximal dendrites elicited oscillations in neuronal calcium concentration (assessed with the calcium indicator Fluo-4) and eventually led to a sustained and lethal increase in intracellular calcium. T cell invasion and effects on neuronal calcium occurred with T cells targeted against a nonmurine antigen, as well as those targeted against proteolipid protein. The same was true of T cells derived from mice whose major histocompatibility complex (MHC) did not match the MHC of the mice providing the brain slice. Calcium overload could be prevented by either pharmacological block of perforin or of either N -methyl-D-aspartate or α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid-kainate type glutamate receptors, which suggests that glutamate toxicity and direct cytotoxic effects combined to elicit neuronal death. R. Nitsch, E. E. Pohl, A. Smorodchenko, C. Infante-Duarte, O. Aktas, F. Zipp, Direct impact of T cells on neurons revealed by two-photon microscopy in living brain tissue. J. Neurosci . 24 , 2458-2464 (2004). [Abstract] [Full Text]

The relationship between neuronal calcium concentration and firing rate during stochastic synaptic inputs

We propose a novel, nonlinear theory about reading neuronal information using intracellular calcium concentrations, which includes the linear theory already developed in the literature as a special case. The theory is numerically confirmed using the Pinsky–Rinzel and integrate-and-fire models with constant rate Poisson inputs. Applying the theory to models with non-constant inputs, we find that there is a time lag equal to the calcium buffering time constant between the instantaneous firing rate and the firing rate estimated using calcium concentrations.