Development of depolarization-induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K(+) concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K(+) (1 min) with a transient increase in [Ca(2+)](i) of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K(+), the amplitude of the [Ca(2+)](i) transients averaging 592 nM. At later stages, all cells responded to 25 mM K(+) with [Ca(2+)](i) transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca(2+)](i) transients. The amplitudes of these [Ca(2+)](i) transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca(2+)](i) transients were greatly reduced in Ca(2+)-free, EGTA-buffered saline, and in the presence of the Ca(2+) channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca(2+) influx through voltage-activated Ca(2+) channels into antennal lobe glial cells. The development of the depolarization-induced Ca(2+) transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna.     

Astrocytes mediate HIV‐1 Tat‐induced neuronal damage via ligand‐gated ion channel P2X7R

During human immunodeficiency virus (HIV)‐1 infection, perturbations in neuron–glia interactions may culminate in neuronal damage. Recently, purinergic receptors have been implicated in the promotion of virus‐induced neurotoxicity and supporting the viral life cycle at multiple stages. The astrocytes robustly express purinergic receptors. We therefore sought to examine if P2X7R, a P2X receptor subtype, can mediate HIV‐1 Tat‐induced neuronal apoptosis. Tat augmented the expression of P2X7R in astrocytes. Our data reveal the involvement of P2X7R in Tat‐mediated release of monocyte chemoattractant protein (MCP‐1) /chemokine (C‐C motif) ligand 2 (CCL2) from the astrocytes. P2X7R antagonists, such as the oxidized ATP, A438079, brilliant blue G, and broad spectrum P2 receptor antagonist suramin, attenuated Tat‐induced CCL2 release in a calcium‐ and extracellular signal‐regulated kinase (ERK)1/2‐dependent manner. Calcium chelators, (1,2‐bis(o‐aminophenoxy) ethane‐N,N,N',N'‐tetraacetic acid) acetoxymethyl ester and EGTA, and ERK1/2 inhibitor U0126 abolished chemokine (C‐C motif) ligand 2 release from astrocytes. Furthermore, in human neuronal cultures, we demonstrated P2X7R involvement in Tat‐mediated neuronal death. Importantly, in the TUNEL assay, the application of P2X7R‐specific antagonists or the knockdown of P2X7R in human astrocytes reduced HIV‐Tat‐induced neuronal death significantly, underlining the critical role of P2X7R in Tat‐mediated neurotoxicity. Our study provides novel insights into astrocyte‐mediated neuropathogenesis in HIV‐1 infection and a novel target for therapeutic management of neuroAIDS.

     

Calcium‐induced calcium release from intracellular stores is developmentally regulated in primary cultures of cerebellar granule neurons

The properties of depolarization‐evoked calcium transients are known to change during the maturation of dissociated cerebellar granule neuron cultures. Here, we assessed the role of the calcium‐induced calcium release (CICR) mechanism in granule neuron maturation. Both depletion of intracellular calcium stores and the pharmacological blockade of CICR significantly reduced depolarization stimulated calcium transients in young but not older (≥1 week) cultures. This functional decrease in the CICR signaling component was associated with the reduction of ryanodine receptor (RyR) immunoreactivity during granule neuron maturation both in culture and in the intact cerebellum. These observations are consistent with the idea that changes in RyR expression result in functional changes in calcium signaling transients during normal neuronal development in the intact mammalian cerebellum as well as in reduced neuronal cultures. Pharmacological disruption of CICR during neuron differentiation in vitro resulted in dose‐dependent changes in survival, GAP‐43 expression, and the acquisition of the glutamatergic neurotransmitter phenotype. Together, these results indicate that CICR function plays a physiologically relevant role in regulating early granule neuron differentiation in vitro and is likely to play a role in cerebellar maturation. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 134–147, 2000     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.

     

Role of N‐ and L‐type calcium channels in depolarization‐induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals

Multiple types of voltage‐activated calcium (Ca²⁺) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca²⁺ influx through N‐ and L‐ type voltage‐activated Ca²⁺ channels in sympathetic neurons to the depolarization‐induced activation of tyrosine hydroxylase (TH), the rate‐limiting enzyme in norepinephrine (NE) synthesis, and the depolarization‐induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both ω‐conotoxin GVIA (1 μM), a specific blocker of N‐type channels, and nimodipine (1 μM), a specific blocker of L‐type Ca²⁺ channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K⁺, indicating that Ca²⁺ influx through both types of channels contributes to enzyme activation. In contrast, K⁺ stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by ω‐conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K⁺ stimulation of NE release from both ganglia and irises was also blocked completely when ω‐conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca²⁺ influx through N‐ and L‐type Ca²⁺ channels. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 137–148, 1999     

Linkages between nectaring and oviposition preferences of Manduca sexta on two co‐blooming Datura species in the Sonoran Desert

1. The oviposition choices of phytophagous insects determine the environment that their offspring will experience, affecting both larval performance and host plant fitness. These choices, however, may be influenced by other activities, such as nectar foraging. 2. In the Sonoran Desert, Manduca sexta (Lepidoptera: Sphingidae) oviposits primarily on the perennial herb Datura wrightii. It has been reported to oviposit on the smaller‐flowered, co‐blooming, sympatric annual Datura discolor as well. Datura is also M. sexta's most important source of nectar in this region. Nectaring and oviposition decisions thus determine M. sexta's reproductive success, as well as the relative benefits (pollination) and costs (herbivory) that each Datura species derives from this interaction. 3. The nectaring and oviposition choices of adult M. sexta on these congeners were studied to investigate how nectar foraging influences oviposition. Larval performance on the two hosts was then assessed. 4. Nectaring and oviposition were behaviourally linked, with M. sexta preferring the perennial D. wrightii as both a nectar source and larval host when given a choice between the two species. This preference disappeared, however, when only D. discolor bore flowers. 5. In the laboratory, larvae developed at equal rates when fed D. wrightii or D. discolor leaves, but survival was higher on D. wrightii when larvae fed on intact plants. 6. These findings suggest that while female preferences match larval performance in most cases, the link between nectaring and oviposition may at times bias moths to lay eggs on inferior larval hosts.     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.     

Brain injury‐induced proteolysis is reduced in a novel calpastatin‐overexpressing transgenic mouse

The calpain family of calcium‐dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene‐knockout strategies have targeted calpains to determine their contribution to neurodegenerative pathology; however, limitations associated with treatment paradigms, drug specificity, and genetic disruptions have produced inconsistent results and complicated interpretation. Specific, targeted calpain inhibition achieved by enhancing endogenous calpastatin levels offers unique advantages in studying pathological calpain activation. We have characterized a novel calpastatin‐overexpressing transgenic mouse model, demonstrating a substantial increase in calpastatin expression within nervous system and peripheral tissues and associated reduction in protease activity. Experimental activation of calpains via traumatic brain injury resulted in cleavage of α‐spectrin, collapsin response mediator protein‐2, and voltage‐gated sodium channel, critical proteins for the maintenance of neuronal structure and function. Calpastatin overexpression significantly attenuated calpain‐mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury, but with no effect on acute hippocampal neurodegeneration. Augmenting calpastatin levels may be an effective method for calpain inhibition in traumatic brain injury and neurodegenerative disorders.     

Domestication of tomato has reduced the attraction of herbivore natural enemies to pest‐damaged plants

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Selective pressure modulation of synaptic voltage‐dependent calcium channels—involvement in HPNS mechanism

Exposure to hyperbaric pressure (HP) exceeding 100 msw (1.1 MPa) is known to cause a constellation of motor and cognitive impairments named high‐pressure neurological syndrome (HPNS), considered to be the result of synaptic transmission alteration. Long periods of repetitive HP exposure could be an occupational risk for professional deep‐sea divers. Previous studies have indicated the modulation of presynaptic Ca²⁺ currents based on synaptic activity modified by HP. We have recently demonstrated that currents in genetically identified cellular voltage‐dependent Ca²⁺ channels (VDCCs), Ca_V1.2 and Ca_V3.2 are selectively affected by HP. This work further elucidates the HPNS mechanism by examining HP effect on Ca²⁺ currents in neuronal VDCCs, Ca_V2.2 and Ca_V2.1, which are prevalent in presynaptic terminals, expressed in Xenopus oocytes. HP augmented the Ca_V2.2 current amplitude, much less so in a channel variation containing an additional modulatory subunit, and had almost no effect on the Ca_V2.1 currents. HP differentially affected the channels' kinetics. It is, therefore, suggested that HPNS signs and symptoms arise, at least in part, from pressure modulation of various VDCCs.     

Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999     

nNOS–CAPON interaction mediates amyloid‐β‐induced neurotoxicity,especially in the early stages

In neurons, increased protein–protein interactions between neuronal nitric oxide synthase (nNOS) and its carboxy‐terminal PDZ ligand (CAPON) contribute to excitotoxicity and abnormal dendritic spine development, both of which are involved in the development of Alzheimer's disease. In models of Alzheimer's disease, increased nNOS–CAPON interaction was detected after treatment with amyloid‐β in vitro, and a similar change was found in the hippocampus of APP/PS1 mice (a transgenic mouse model of Alzheimer's disease), compared with age‐matched background mice in vivo. After blocking the nNOS–CAPON interaction, memory was rescued in 4‐month‐old APP/PS1 mice, and dendritic impairments were ameliorated both in vivo and in vitro. Furthermore, we demonstrated that S‐nitrosylation of Dexras1 and inhibition of the ERK–CREB–BDNF pathway might be downstream of the nNOS–CAPON interaction.     

Spike‐dependent calcium influx in dendrites of the cricket giant interneuron

Identified wind‐sensitive giant interneurons in the cricket's cercal sensory system integrate cercal afferent signals and release an avoidance behavior. A calcium‐imaging technique was applied to the giant interneurons to examine the presence of the voltage‐dependent Ca²⁺ channels (VDCCs) in their dendrites. We found that presynaptic stimuli to the cercal sensory nerve cords elevated the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the dendrites of the giant interneurons. The dendritic Ca²⁺ rise coincided with the spike burst of the giant interneurons, and the rate of Ca²⁺ rise depended on the frequency of the action potentials. These results suggest that the action potentials directly caused [Ca²⁺]_i increase. Observation of the [Ca²⁺]_i elevation induced by depolarizing current injection demonstrates the presence of the VDCCs in the dendrites. Although hyperpolarizing current injection into the giant interneuron suppressed action potential generation, EPSPs could induce no [Ca²⁺]_i increase. This result means that ligand‐gated channels do not contribute to the synaptically stimulated Ca²⁺ elevation. On the other hand, antidromically stimulated spikes also increased [Ca²⁺]_i in all cellular regions including the dendrites. And bath application of a mixture of Ni²⁺, Co²⁺, and Cd²⁺ or tetrodotoxin inhibited the [Ca²⁺]_i elevation induced by the antidromic stimulation. From these findings, we suppose that the axonal spikes antidromically propagate and induce the Ca²⁺ influx via VDCCs in the dendrites. The spike‐dependent Ca²⁺ elevation may regulate the sensory signals processing via second‐messenger cascades in the giant interneurons. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 45–56, 2000     

Influx of calcium through L‐type calcium channels in early postnatal regulation of chloride transporters in the rat hippocampus

During the early postnatal period, GABA_B receptor activation facilitates L‐type calcium current in rat hippocampus. One developmental process that L‐type current may regulate is the change in expression of the K⁺Cl^? co‐transporter (KCC2) and N⁺K⁺2Cl^? co‐transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L‐type current, GABA_B receptors, and expression of chloride transporters during development. The facilitation of L‐type current by GABA_B receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L‐type channel antagonist and a GABA_B receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L‐type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L‐type channels in the early postnatal period in hippocampal neurons. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

小檗碱对豚鼠结肠平滑肌细胞内游离钙浓度的影响

采用Ca2 荧光示踪剂Fura 2 AM和双波长荧光分光光度法 ,观察小檗碱 (berberine ,Ber)对酶法分离的豚鼠结肠平滑肌细胞内游离钙 ([Ca2 ]i)的影响并探讨其机制。在含 1 5mmol/LCaCl2 的HEPES Ringer缓冲液中 ,豚鼠结肠平滑肌细胞 [Ca2 ]i为 10 8± 9 4nmol/L (n =7) ,Ber对静息 [Ca2 ]i 无明显影响 ,Ber呈浓度依赖性抑制 ,6 0mmol/LKCl引起的 [Ca2 ]i 增高 ,IC50 值为 34 0 9μmol/L。在含 1 5mmol/LCa2 和无Ca2 的缓冲液中 ,30、10 0μmol/LBer均显著抑制 10 μmol/LACh所诱发的 [Ca2 ]i 的增高 ,且有浓度依赖性 ;同样Ber对环匹阿尼酸 (CPA)所致的 [Ca2 ]i 增高也有浓度依赖性抑制作用 ,有钙和无钙条件下IC50 分别为 37 97μmol/L和 49 70 μmol/L。结果提示 ,Ber对结肠平滑肌细胞外Ca2 内流和细胞内钙释放均有抑制作用。     

Aquatic insect β‐diversity is not dependent on elevation in Southern Rocky Mountain streams

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