Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus.

Presynaptic inhibition of neurotransmitter release is thought to be mediated by a reduction of axon terminal Ca2+ current. We have compared the actions of several known inhibitors of evoked glutamate release with the actions of the Ca2+ channel antagonist Cd2+ on action potential-independent synaptic currents recorded from CA3 neurons in hippocampal slice cultures. Baclofen and adenosine decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Cd2+ blocked evoked synaptic transmission, but had no effect on the frequency or amplitude of either mEPSCs or inhibitory postsynaptic currents (IPSCs). Inhibition of presynaptic Ca2+ current therefore appears not to be required for the inhibition of glutamate release by adenosine and baclofen. Baclofen had no effect on the frequency of miniature IPSCs, indicating that gamma-aminobutyric acid B-type receptors exert distinct presynaptic actions at excitatory and inhibitory synapses.     

Simulation of postsynaptic glutamate receptors reveals critical features of glutamatergic transmission

Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following high-frequency stimulation. They also provide a new tool to analyze the interactions between metabotropic and ionotropic glutamate receptors.     

Use of Calcium Antagonism for the Characterization of Drug-Evoked Dopamine Release from the Brain of Conscious Rats Determined by Microdialysis

Dopamine was determined by microdialysis of the striatum of conscious rats. We investigated whether the release of dopamine, induced by nine different pharmacological treatments, was sensitive to calcium antagonism. Calcium antagonism was determined by Mg2+ or Cd2+ infusion. The following conditions were investigated: haloperidol, haloperidol plus GBR 12909, nomifensine, (+)-amphetamine (all administered intraperitoneally), KCl, 1-methyl-4-phenyl-pyridinium ion (MPP+), glutamate, ouabain, and 120 mmol/L magnesium (all applied by infusion through the dialysis membrane). The results on calcium antagonism were combined with data on tetrodotoxin (TTX) sensitivity. With the combined data, three different types of dopamine release were characterized. First, action potential-dependent dopamine release was observed in animals treated with saline, haloperidol, haloperidol plus GBR 12909, nomifensine, and ouabain. Second, action potential-independent release was established in the case of (+)-amphetamine, glutamate, MPP+, and 120 mmol/L Mg2+. Finally, K+-induced dopamine release was classified as TTX independent and calcium dependent. It is concluded that brain dialysis is a powerful method for differentiating between different types of neurotransmitter release.     

Kainic acid and synaptic transmission in the stellate ganglion of the squid.

Kainate, a conformational analogue of glutamate, blocks synaptic transmission across the giant synapse of the squid. In the presence of blocking doses of kainate, impulses continue to propagate into the nerve terminal, but action potentials are slightly reduced in size and the subsequent hyperpolarization is greatly diminished. Kainate depolarizes the postsynaptic axon. Since the depolarizing action of kainate is confined to the postsynaptic membrane, it appears that kainate can combine with the receptors which are normally activated by the transmitter. This results in a diminished effect of the transmitter released by a presynaptic nerve impulse.     

Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways

Functional neuroimaging uses activity-dependent changes in cerebral blood flow to map brain activity, but the contributions of presynaptic and postsynaptic activity are incompletely understood, as are the underlying cellular pathways. Using intravital multiphoton microscopy, we measured presynaptic activity, postsynaptic neuronal and astrocytic calcium responses, and erythrocyte velocity and flux in olfactory glomeruli during odor stimulation in mice. Odor-evoked functional hyperemia in glomerular capillaries was highly correlated with glutamate release, but did not require local postsynaptic activity. Odor stimulation induced calcium transients in astrocyte endfeet and an associated dilation of upstream arterioles. Calcium elevations in astrocytes and functional hyperemia depended on astrocytic metabotropic glutamate receptor 5 and cyclooxygenase activation. Astrocytic glutamate transporters also contributed to functional hyperemia through mechanisms independent of calcium rises and cyclooxygenase activation. These local pathways initiated by glutamate account for a large part of the coupling between synaptic activity and functional hyperemia in the olfactory bulb.     

A realistic model of biphasic calcium transients in electrically nonexcitable cells.

In many electrically nonexcitable cells, the release of calcium from internal stores is followed by a much slower phase in which the intracellular calcium concentration decreases gradually to a sustained value higher than the concentration before stimulation. This elevated calcium plateau has been shown to be the result of calcium influx. The model presented in this work describes a system consisting of a cytoplasmic calcium store and a plasma membrane calcium channel, both excitable by a membrane receptor; a fast cytoplasmic calcium buffer; and calcium pumps in both the calcium store and cellular membranes. Inherent difficulties in the numerical evaluation of the model, caused by very large calcium fluxes across the store membrane, were overcome by analytically separating the fast processes of calcium release from the slower processes of calcium cycling across the plasma membrane. This enabled the simulation of realistic biphasic calcium transients similar to those observed experimentally. The model predicted 1) a strong correlation between the rate of calcium cycling across the plasma membrane and the rate of calcium decay; and 2) a dependence on the level of cell excitation of the maximum rise in cytoplasmic calcium concentration, the level of the elevated calcium plateau, and the rate of calcium decay. Using the model, we simulated the washout of agonist from the bathing solution and the depletion of the calcium store by a pharmacological agent (such as thapsigargin) under several experimental conditions.     

Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons.

Opiates and the opioid peptide enkephalin can cause indirect excitation of principal cortical neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons. The mechanism by which opioids mediate these effects on interneurons is unknown, but enkephalin hyperpolarizes the somatic membrane potential of a variety of neurons in the brain, including hippocampal interneurons. We now report a new, more direct mechanism for the opioid-mediated reduction in synaptic inhibition. The enkephalin analog D-Ala2-Met5-enkephalinamide (DALA) decreases the frequency of miniature, action potential-independent, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) without causing a change in their amplitude. Thus, we conclude that DALA inhibits the action potential-independent release of GABA through a direct action on interneuronal synaptic terminals. In contrast, DALA reduces the amplitude of action potential-evoked, GABA-mediated IPSCs, as well as decreases their frequency. This suggests that the opioid-mediated inhibition of non-action potential-dependent GABA release reveals a mechanism that contributes to reducing action potential-evoked GABA release, thereby decreasing synaptic inhibition.     

Postfusional control of quantal current shape

Whether glutamate is released rapidly, in an all-or-none manner, or more slowly, in a regulated manner, is a matter of debate. We analyzed the time course of excitatory postsynaptic currents (EPSCs) at glutamatergic neuromuscular junctions of Drosophila and found that the decay phase of EPSCs was protracted to a variable extent. The protraction was more pronounced in evoked and spontaneous quantal EPSCs than in action potential-evoked multiquantal EPSCs; reduced in quantal EPSCs from endophilin null mutants, which maintain release via kiss-and-run; and dependent on synaptotagmin isoform, calcium, and protein phosphorylation. Our data indicate that glutamate is released from individual synaptic vesicles for milliseconds through a fusion pore. Quantal glutamate discharge time course depends on presynaptic calcium inflow and the molecular composition of the release machinery.     

EFFECT OF STIMULATION OF EXCITATORY NERVE TRACT ON RELEASE OF GLUTAMIC ACID FROM OLFACTORY CORTEX SLICES IN VITRO

Abstract— Thin sections prepared from the olfactory cortex of the guinea pig were incubated in a medium containing [¹⁴C]glutamate, and release of radioactive compounds and electrical activity were subsequently examined in the presence of l -cysteate. The postsynaptic potential was almost completely suppressed in the medium containing l -cysteate, whereas the presynaptic potential was unaffected. Repetitive stimulation of the excitatory input of the lateral olfactory tract enhanced release of radioactive glutamate. The facilitatory effect of lateral olfactory tract stimulation increased with increase in stimulus frequency and was dependent on calcium. Release of radioactive gluiamine was not enhanced by lateral olfactory tract stimulation. Phenobarbitone sodium markedly depressed both the postsynaptic potential and the effect of lateral olfactory tract stimulation on glutamate release. These results indicate that stimulation to the lateral olfactory tract enhances liberation of glutamate from the tract nerve terminals.     

Stores not just for storage. intracellular calcium release and synaptic plasticity.

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.     

Glutamate release and neuronal damage in ischemia.

Neuronal injury caused by ischemia after occlusion of cerebral arteries is believed to be mediated by excessive activation of glutamate receptors. In the ischemic brain, extracellular glutamate is elevated rapidly after the onset of ischemia and declines following reperfusion. The mechanisms of the elevation of extracellular glutamate include enhanced efflux of glutamate and the reduction of glutamate uptake. The early efflux of glutamate occurring immediately after the onset of ischemia is mediated by a calcium-dependent process through activation of voltage-dependent calcium channels. The calcium-independent efflux at later stages is thought to be mediated primarily by glutamate transporters operating in the reverse mode owing to the imbalance of sodium ions across plasma membranes. Although high levels of glutamate in the extracellular space are well established to appear rapidly after the onset of ischemia, a direct linkage between the enhanced release of glutamate and the neuronal injury has not been fully established. In cultured neurons, ischemia induces efflux of glutamate into the extracellular space, but subsequent neuronal loss is not solely caused by the high glutamate concentration. In addition, cultured neurons can be rescued by NMDA antagonists added to the medium after exposure to glutamate receptor agonists. Two mechanisms can be proposed for neuroprotection by late NMDA receptor blockade, i.e., blocking of presynaptic release of glutamate after excessive activation of glutamate receptors, and blocking of postsynaptic sensitization of NMDA receptors.     

Fast inhibition of glutamate-activated currents by caffeine

Background

Caffeine stimulates calcium-induced calcium release (CICR) in many cell types. In neurons, caffeine stimulates CICR presynaptically and thus modulates neurotransmitter release.

Methodology/Principal Findings

Using the whole-cell patch-clamp technique we found that caffeine (20 mM) reversibly increased the frequency and decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) in neocortical neurons. The increase in mEPSC frequency is consistent with a presynaptic mechanism. Caffeine also reduced exogenously applied glutamate-activated currents, confirming a separate postsynaptic action. This inhibition developed in tens of milliseconds, consistent with block of channel currents. Caffeine (20 mM) did not reduce currents activated by exogenous NMDA, indicating that caffeine block is specific to non-NMDA type glutamate receptors.

Conclusions/Significance

Caffeine-induced inhibition of mEPSC amplitude occurs through postsynaptic block of non-NMDA type ionotropic glutamate receptors. Caffeine thus has both pre and postsynaptic sites of action at excitatory synapses.     

Calmodulin activation by calcium transients in the postsynaptic density of dendritic spines

The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM) and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.     

The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.     

Mechanism of store-operated calcium entry

Activation of receptors coupled to the phospholipase C/IP₃ signalling pathway results in a rapid release of calcium from its intracellular stores, eventually leading to depletion of these stores. Calcium store depletion triggers an influx of extracellular calcium across the plasma membrane, a mechanism known as the store-operated calcium entry or capacitative calcium entry. Capacitative calcium current plays a key role in replenishing calcium stores and activating various physiological processes. Despite considerable efforts, very little is known about the molecular nature of the capacitative channel and the signalling pathway that activates it. This review summarizes our current knowledge about store operated calcium entry and suggests possible hypotheses for its mode of activation.     

Mechanisms for regulating synaptic efficiency in the visual cortex

Brief epochs of pairing of low frequency synaptic activation and postsynaptic depolarization, in vitro, in supragranular neurons of mature guinea-pig visual cortex lead to a transient (20–60 min) synaptic potentiation. This process is due to a true up-regulation of excitatory synapse efficiency onto the activated neuron. The potentiation requires NMDA receptor activation and a postsynaptic calcium signal for induction and it is modifiable by endogenous nitric oxide (NO) production in the mature cortex. In the cortex of young animals (< PND 21), the pairing-induced potentiation is robust and depends on a postsynaptic calcium signal but it is independent of NMDA receptor activation and NO production. The ability of cortical synaptosomes to release endogenous glutamate is enhanced by NMDA receptor activation and this enhancement is NO-dependent. The NO signal, however, does not amplify the glutamate release of all synapses but only those that have activated voltage-gated calcium channels and were presumably more active at the time of the NO signal. Electrophysiological recordings from visual cortical neurons in anesthetized cats with local iontophoresis of compounds that inhibit or facilitate endogenous cortical NO production reveal the capacity for NO to modulate visual responses in vivo. NO appears to act in the intact cortex by amplifying signals of visual inputs that were co-active at the time of the NO production. The adult visual cortex is capable of dramatic alterations in synaptic efficiency over brief periods suggesting a dynamic cortical network. NMDA receptors and nitric oxide contribute to these processes.     

Evidence for postsynaptic modulation of muscle contraction by a Drosophila neuropeptide

DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10(-8)M. The increase in tonus persisted in the presence of 7x10(-3)M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.     

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity

The lethal toxin ofBacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neuroprotective Effect of Hypothermia in Cortical Cultures Exposed to Oxygen-Glucose Deprivation or Excitatory Amino Acids

Abstract: We examined the effect of moderate hypothermia (30°C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.     

Endocannabinoids control the induction of cerebellar LTD

The long-term depression (LTD) of parallel fiber (PF) synapses onto Purkinje cells plays a central role in motor learning. Endocannabinoid release and LTD induction both depend upon activation of the metabotropic glutamate receptor mGluR1, require postsynaptic calcium increases, are synapse specific, and have a similar dependence on the associative activation of PF and climbing fiber synapses. These similarities suggest that endocannabinoid release could account for many features of cerebellar LTD. Here we show that LTD induction is blocked by a cannabinoid receptor (CB1R) antagonist, by inhibiting the synthesis of the endocannabinoid 2-arachidonyl glycerol (2-AG), and is absent in mice lacking the CB1R. Although CB1Rs are prominently expressed presynaptically at PF synapses, LTD is expressed postsynaptically. In contrast, a previously described transient form of inhibition mediated by endocannabinoids is expressed presynaptically. This indicates that Purkinje cells release 2-AG that activates CB1Rs to both transiently inhibit release and induce a postsynaptic form of LTD.