The role of perisynaptic glial sheaths in glutamate spillover and extracellular Ca(2+) depletion

Recent findings suggest that rapid activation of extrasynaptic receptors and transient depletion of extracellular Ca(2+) may represent an important component of glutamatergic synaptic transmission. These phenomena imply a previously unrecognized role for synaptic glial sheaths: to retard extracellular diffusion in the synaptic vicinity. The present study is an attempt to assess the extent and physiological implications of this retardation using a detailed compartmental model of the typical synaptic environment. The model allows reconstruction of a partial (asymmetric) glial sheath covered with transporter molecules, which gives a more realistic representation of the vicinity of central synapses. Simulations show to what extent, in conditions compatible with physiology, the occupancy of synaptic receptors and the depletion of Ca(2+) in the cleft increase with increased glial coverage. The impact of glial sheaths on synaptic transmission is shown to become greater with smaller synapses and with slower kinetics of perisynaptic ion transients. At a calyceal synapse, a profound temporal filtering of fast Ca(2+) influx is found, and similar phenomena are predicted to occur following simultaneous activation of multiple synapses in the neuropil. The results provide a quantitative guidance for interpretation of physiological experiments that address fast transients of neurotransmitters and small ions in the brain tissue.     

The life cycle of Ca(2+) ions in dendritic spines

Spine Ca(2+) is critical for the induction of synaptic plasticity, but the factors that control Ca(2+) handling in dendritic spines under physiological conditions are largely unknown. We studied [Ca(2+)] signaling in dendritic spines of CA1 pyramidal neurons and find that spines are specialized structures with low endogenous Ca(2+) buffer capacity that allows large and extremely rapid [Ca(2+)] changes. Under physiological conditions, Ca(2+) diffusion across the spine neck is negligible, and the spine head functions as a separate compartment on long time scales, allowing localized Ca(2+) buildup during trains of synaptic stimuli. Furthermore, the kinetics of Ca(2+) sources governs the time course of [Ca(2+)] signals and may explain the selective activation of long-term synaptic potentiation (LTP) and long-term depression (LTD) by NMDA-R-mediated synaptic Ca(2+).     

The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C

Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.     

CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.     

Zinc signaling in the hippocampus and its relation to pathogenesis of depression

Histochemically reactive zinc (Zn(2+)) is co-released with glutamate from zincergic neurons, a subclass of glutamatergic neurons. Zn(2+) serves as a signal factor in both the extracellular and intracellular compartments. Glucocorticoid-glutamatergic interactions have been proposed as a potential model to explain stress-mediated impairment of hippocampal function, i.e., cognition. However, it is unknown whether glucocorticoid-zincergic interactions are involved in this impairment. In the present study, involvement of synaptic Zn(2+) in stress-induced attenuation of CA1 LTP was examined in hippocampal slices from young rats after exposure to tail suspension stress for 30s, which significantly increased serum corticosterone. Stress-induced attenuation of CA1 LTP was ameliorated by administration of clioquinol, a membrane permeable zinc chelator, to rats prior to exposure to stress, implying that the reduction of synaptic Zn(2+) by clioquinol participates in this amelioration. To pursue the involvement of corticosterone-mediated Zn(2+) signal in the attenuated CA1 LTP by stress, dynamics of synaptic Zn(2+) was checked in hippocampal slices exposed to corticosterone. Corticosterone increased extracellular Zn(2+) levels measured with ZnAF-2 dose-dependently, as well as the intracellular Ca(2+) levels measured with calcium orange AM, suggesting that corticosterone excites zincergic neurons in the hippocampus and increases Zn(2+) release from the neuron terminals. Intracellular Zn(2+) levels measured with ZnAF-2DA were also increased dose-dependently, but not in the coexistence of CaEDTA, a membrane-impermeable zinc chelator, suggesting that intracellular Zn(2+) levels is increased by the influx of extracellular Zn(2+). Furthermore, corticosterone-induced attenuation of CA1 LTP was abolished in the coexistence of CaEDTA. The present study suggests that corticosterone-mediated increase in postsynaptic Zn(2+) signal in the cytosolic compartment is involved in the attenuation of CA1 LTP after exposure to acute stress. We propose that corticosterone-mediated increase in postsynaptic Zn(2+) signal, which is induced by acute stress, changes hippocampal function and then is possibly a risk factor under chronic stress circumstances to induce depressive symptoms.     

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.     

Ca2+ signalling in postsynaptic dendrites and spines of mammalian neurons in brain slice.

Postsynaptic Ca2+ changes are involved in control of cellular excitability and induction of synaptic long-term changes. We monitored Ca2+ changes in dendrites and spines during synaptic and direct stimulation using high resolution microfluorometry of fura-2 injected into CA3 pyramidal neurons in guinea pig hippocampal slice. When driven by current injection from an intracellular electrode or with synaptic stimulation, postsynaptic Ca2+ accumulations were highest in the proximal dendrites with a pronounced fall-off towards the soma and some fall-off towards more distal dendrites. Muscarinic activation by low concentrations of carbachol strongly increased intradendritic Ca2+ accumulation during directly-evoked repetitive firing. This enhancement occurred in large part because muscarinic activation suppressed the normal Ca(2+)-dependent activation of K-channels that mediates adaptation of firing. Repetitive firing of cholinergic fibers in the slice reproduced the effects of carbachol. Inhibition of acetylcholine-esterase activity by eserine enhanced the effects of repetitive stimulation of chlolinergic fibers. All effects were reversible and were blocked by the muscarinic antagonist atropine. Ca2+ accumulations in postsynaptic spines might be the basis of specificity of synaptic plasticity. With selective stimulation of few associative/comissural fibers, Ca2+ accumulated in single postsynaptic spines but not in the parent dendrite. With strong stimulation, dendrite levels also increased but spine levels were considerably higher. The NMDA-receptor antagonist AP-5 blocked Ca(2+)-peaks in spines, but left Ca2+ changes in dendrite shafts largely unaffected. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, developed with repeated stimulation. Our results demonstrate a spine entity that can act independent from the dendrite with respect to Ca(2+)-dependent processes. Muscarinic augmentation of dendritic Ca2+ levels might reduce diffusional loss of Ca2+ from hot spines into the parent dendrite, thus supporting cooperativity and associativity of synaptic plasticity.     

Assessment of frequency-dependent alterations in the level of extracellular Ca2+ in the synaptic cleft.

The synaptic cleft may be represented as a very thin disk of extracellular fluid. It is possible that at high stimulation frequencies the interval between pulses would be insufficient for diffusion of Ca2+ from the periphery of the cleft to replace extracellular Ca2+ depleted at the center of the cleft as a result of activation of postsynaptic, Ca2(+)-permeable channels. Computer modeling was employed to assess the impact of activation of glutamate receptor channels (GRCs) in the postsynaptic membrane on the level of extracellular Ca2+ within the synaptic cleft. The model includes calcium influx from the synaptic cleft into the postsynaptic compartment through GRC and calcium efflux through calcium pumps and Na/Ca exchangers. Concentrations of extracellular Ca2+ inside the cleft are estimated by using a compartmental model incorporating flux across the postsynaptic membrane and radial diffusion from the edges of the cleft. The simulations suggest that substantial extracellular Ca2+ depletion can occur in the clefts during activation of GRCs, particularly at high stimulation frequencies used to induce long-term potentiation (LTP). Only minimal transitory changes in extracellular Ca2+ are observed at low frequencies. These frequency-dependent alterations in extracellular Ca2+ dynamics are a direct reflection of the activity of GRCs and could be involved in the modulation of presynaptic function via a retrograde messenger mechanism, if there are extracellular Ca2+ sensors on the presynaptic membranes. The recently cloned extracellular Ca2(+)-sensing receptors that are known to be present in nerve terminals in hippocampus and other areas of the brain could potentially play such a role.     

Cholinergic input uncouples Ca2+ changes from K+ conductance activation and amplifies intradendritic Ca2+ changes in hippocampal neurons

Muscarinic synaptic activation is known to be involved in cortical arousal as well as learning. Although simple increases in the electrical responsiveness of neurons might be the basis of arousal, the linkage of muscarinic transmission to the synaptic plasticity that might underlie learning is lacking. Most models of synaptic plasticity involve postsynaptic Ca2+ changes as a trigger for subsequent processes. We imaged muscarinic effects on free Ca2+ accumulation during intracellular recordings from CA3 pyramidal neurons in the guinea pig hippocampal slice. Muscarinic activation, either by repetitive stimulation of cholinergic fibers or by bath-applied carbachol, strongly increased intradendritic Ca2+ accumulation during directly evoked repetitive firing, in part by blocking a Ca(2+)-dependent K+ conductance. The effects of repetitive stimulation of cholinergic fibers were enhanced by the acetylcholine-esterase blocker eserine and blocked by the muscarinic antagonist atropine. These findings demonstrate a novel muscarinic reinforcement of Ca2+ changes during excitation, which are probably significant for synapse modification.     

An investigation into signal transduction mechanisms involved in insulin-induced long-term depression in the CA1 region of the hippocampus

Recent work has demonstrated that brief application of insulin to hippocampal slices can induce a novel form of long-term depression (insulin-LTD) in the CA1 region of the hippocampus; however, the molecular details of how insulin triggers LTD remain unclear. Using electrophysiological and biochemical approaches in the hippocampal slices, we show here that insulin-LTD (i) is specific to 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor- but not NMDA receptor-mediated synaptic transmission; (ii) is induced and expressed postsynaptically but does not require the activation of ionotropic and metabotropic glutamate receptors; (iii) requires a concomitant Ca(2+) influx through l-type voltage-activated Ca(2+) channels (VACCs) and the release of Ca(2+) from intracellular stores; (iv) requires the series of protein kinases, including protein tyrosine kinase (PTK), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC); (v) is mechanistically distinct from low-frequency stimulation-induced LTD (LFS-LTD) and independent on protein phosphatase 1/2 A (PP1/2 A) and PP2B activation; (vi) is dependent on a rapamycin-sensitive local translation of dendritic mRNA, and (vii) is associated with a persistent decrease in the surface expression of GluR2 subunit. These results suggest that a PI3K/PKC-dependent insulin signaling, which controls postsynaptic surface AMPA receptor numbers through PP-independent endocytosis, may be a major expression mechanism of insulin-LTD in hippocampal CA1 neurons.     

Involvement of glucocorticoid-mediated Zn2+ signaling in attenuation of hippocampal CA1 LTP by acute stress

Glucocorticoid-glutamatergic interactions have been proposed as a potential model to explain stress-mediated impairment of cognition. However, it is unknown whether glucocorticoid-zincergic interactions are involved in this impairment. Histochemically reactive zinc (Zn(2+)) is co-released with glutamate from zincergic neurons. In the present study, involvement of synaptic Zn(2+) in stress-induced attenuation of CA1 LTP was examined in hippocampal slices from young rats after exposure to tail suspension stress for 30s, which significantly increased serum corticosterone. Stress-induced attenuation of CA1 LTP was ameliorated by administration of clioquinol, a membrane permeable zinc chelator, to rats prior to exposure to stress, implying that the reduction of synaptic Zn(2+) by clioquinol participates in this amelioration. To pursue the involvement of corticosterone-mediated Zn(2+) signal in the attenuated CA1 LTP by stress, dynamics of synaptic Zn(2+) was checked in hippocampal slices exposed to corticosterone. Corticosterone increased extracellular Zn(2+) levels measured with ZnAF-2 dose-dependently, as well as the intracellular Ca(2+) levels measured with calcium orange AM, suggesting that corticosterone excites zincergic neurons in the hippocampus and increases Zn(2+) release from the neuron terminals. Intracellular Zn(2+) levels measured with ZnAF-2DA were also increased dose-dependently, but not in the coexistence of CaEDTA, a membrane-impermeable zinc chelator, suggesting that intracellular Zn(2+) levels is increased by the influx of extracellular Zn(2+). Furthermore, corticosterone-induced attenuation of CA1 LTP was abolished in the coexistence of CaEDTA. The present study suggests that corticosterone-mediated increase in postsynaptic Zn(2+) signal in the cytosolic compartment is involved in the attenuation of CA1 LTP after exposure to acute stress.     

Modulation of hippocampal calcium signalling and plasticity by serine/threonine protein phosphatases

Kinases and phosphatases act antagonistically to maintain physiological phosphorylation/dephosphorylation at numerous intracellular sites critical for neuronal signalling. In this study, it was found that inhibition of serine/threonine phosphatases by exposure of hippocampal slices to okadaic acid (OA) or cantharidin (CA; 100 nmol/L) for 2 h resulted in reduced basal synaptic transmission and blocked the induction of synaptic plasticity in the form of long-term potentiation as determined by electrophysiological analysis. Fura-2 Ca(2+) imaging revealed a bidirectional modulation of N-methyl-D-aspartate (NMDA) -mediated Ca(2+) responses and reduced KCl-mediated Ca(2+) responses in neonatal cultured hippocampal neurons after phosphatase inhibition. While OA inhibited NMDA-induced Ca(2+) influx both acutely and after incubation, CA-enhanced receptor-mediated Ca(2+) signalling at low concentrations (1 nmol/L) but reduced NMDA and KCl-mediated Ca(2+) responses at higher concentrations (100 nmol/L). Changes in Ca(2+) signalling were accompanied by increased phosphorylation of cytoskeletal proteins tau and neurofilament and the NMDA receptor subunit NR1 in selective treatments. Incubation with OA (100 nmol/L) also led to the disruption of the microtubule network. This study highlights novel signalling effects of prolonged inhibition of protein phosphatases and suggests reduced post-synaptic signalling as a major mechanism for basal synaptic transmission and long-term potentiation impairments.     

A D2 class dopamine receptor transactivates a receptor tyrosine kinase to inhibit NMDA receptor transmission

Receptor tyrosine kinases (RTKs) are membrane spanning proteins with intrinsic kinase activity. Although these receptors are known to be involved in proliferation and differentiation of cells, their roles in regulating central synaptic transmission are largely unknown. In CA1 pyramidal neurons, activation of D2 class dopamine receptors depressed excitatory transmission mediated by the NMDA subtype of glutamate receptor. This depression resulted from the quinpirole-induced release of intracellular Ca(2+) and enhanced Ca(2+)-dependent inactivation of NMDA receptors. The dopamine receptor-mediated depression was dependent on the "transactivation" of PDGFRbeta. Therefore, RTK transactivation provides a novel mechanism of communication between dopaminergic and glutamatergic systems and might help to explain how reciprocal changes in these systems could be linked to the deficits in cognition, memory, and attention observed in schizophrenia and attention deficit hyperactivity disorder.     

Ca2+ influx through distinct routes controls exocytosis and endocytosis at drosophila presynaptic terminals

Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca(2+). However, it is not known whether Ca(2+) influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, we separately measured exocytosis and endocytosis using FM1-43. In a temperature-sensitive Ca(2+) channel mutant, cacophony(TS2), exocytosis induced by high K(+) decreased at nonpermissive temperatures, while endocytosis remained unchanged. In wild-type larvae, a spider toxin, PLTXII, preferentially inhibited exocytosis, whereas the Ca(2+) channel blockers flunarizine and La(3+) selectively depressed endocytosis. None of these blockers affected exocytosis or endocytosis induced by a Ca(2+) ionophore. Evoked synaptic potentials were depressed regardless of stimulus frequency in cacophony(TS2) at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La(3+) gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibire(ts1), a dynamin mutant, flunarizine or La(3+) inhibited assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function.     

A constitutive, transient receptor potential-like Ca2+ influx pathway in presynaptic nerve endings independent of voltage-gated Ca2+ channels and Na+/Ca2+ exchange

Calcium levels in the presynaptic nerve terminal are altered by several pathways, including voltage-gated Ca(2+) channels, the Na(+)/Ca(2+) exchanger, Ca(2+)-ATPase, and the mitochondria. The influx pathway for homeostatic control of [Ca(2+)](i) in the nerve terminal has been unclear. One approach to detecting the pathway that maintains internal Ca(2+) is to test for activation of Ca(2+) influx following Ca(2+) depletion. Here, we demonstrate that a constitutive influx pathway for Ca(2+) exists in presynaptic terminals to maintain internal Ca(2+) independent of voltage-gated Ca(2+) channels and Na(+)/Ca(2+) exchange, as measured in intact isolated nerve endings from mouse cortex and in intact varicosities in a neuronal cell line using fluorescence spectroscopy and confocal imaging. The Mg(2+) and lanthanide sensitivity of the influx pathway, in addition to its pharmacological and short hairpin RNA sensitivity, and the results of immunostaining for transient receptor potential (TRP) channels indicate the involvement of TRPC channels, possibly TRPC5 and TRPC1. This constitutive Ca(2+) influx pathway likely serves to maintain synaptic function under widely varying levels of synaptic activity.     

Store-operated Ca2+ influx causes Ca2+ release from the intracellular Ca2+ channels that is required for T cell activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.