Possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters.

The postsynaptic actions of some neurotransmitters may be mediated through cyclic nucleotides and cyclic nucleotide-dependent phosphorylation of specific membrane proteins in postsynaptic cells. In addition to providing a molecular basis for the actions of several neurotransmitters and of certain drugs affecting behaviour, the model suggests a mechanism by which neurotransmitter signals may be converted into electrophysiological responses in postsynaptic cells.     

Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells

Many hormones and neurotransmitters evoke Ca2+ release from intracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3) and cyclic adenosine 5'-diphosphate-ribose (cADPR) are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous metabolite of beta-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1-2s) and long-lasting (0.2-1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.     

Long-term potentiation is associated with increases in quantal content and quantal amplitude.

Long-term potentiation (LTP) of synaptic transmission in CA1 neurons of the hippocampus, elicited by the conjunction of presynaptic firing and postsynaptic depolarization, is an important model of plasticity, which may underlie memory storage. Although induction of LTP takes place in the postsynaptic cell, it is not clear whether it is expressed through an enhancement of transmitter release or through an increased postsynaptic response to the same amount of transmitter. Analysis of the trial-to-trial amplitude fluctuations of synaptic signals, that is quantal analysis, gives an important insight into the probabilistic mechanisms of transmission, although attempts to apply it to the mode of expression of LTP have so far yielded inconsistent results, at least in part because they have relied on models of transmitter release that have not been confirmed experimentally. Here we report clear evidence for quantal fluctuation in a subset of cells. Induction of LTP in these cells causes abrupt increases in either quantal content or quantal amplitude, or both. This shows that two different mechanisms can underlie the maintenance of LTP.     

A physiological role for GABAB receptors in the central nervous system

The role of GABA in synaptic transmission in the mammalian central nervous system is more firmly established than for any other neurotransmitter. With virtually every neuron studied, the synaptic action of GABA is mediated by bicuculline-sensitive GABAA receptors which selectively increase chloride conductance. However, it has been shown that GABA has a presynaptic inhibitory action on transmitter release that is insensiive to bicuculline and is selectively mimicked by baclofen. The receptors involved in this action are referred to as GABAB receptors, to distinguish them from the classic bicuculline-sensitive GABAA receptors. In hippocampal pyramidal cells an additional postsynaptic action of GABA and baclofen has been reported that is also insensitive to GABAA antagonists, and may be mediated by GABAB receptors on the postsynaptic neuron. This action of GABA and baclofen involves an increase in potassium conductance. Synaptic activation of pathways converging on hippocampal pyramidal cells results in a slow inhibitory postsynaptic potential which involves an increase in potassium conductance, and it has been suggested that GABAB receptors might be responsible for this synaptic potential. However, to establish convincingly that GABAB receptors are physiologically important in the central nervous system, a selective GABAB antagonist is required. Here we provide this missing evidence. Using the hippocampal slice preparation, we now report that the phosphonic acid derivative of baclofen, phaclofen, is a remarkably selective antagonist of both the postsynaptic action of baclofen and the bicuculline-resistant action of GABA, and that it selectively abolishes the slow inhibitory postsynaptic potential in pyramidal cells.     

Purkinje neuron synchrony elicits time-locked spiking in the cerebellar nuclei

An unusual feature of the cerebellar cortex is that its output neurons, Purkinje cells, release GABA (γ-aminobutyric acid). Their high intrinsic firing rates (50?Hz) and extensive convergence predict that their target neurons in the cerebellar nuclei would be largely inhibited unless Purkinje cells pause their spiking, yet Purkinje and nuclear neuron firing rates do not always vary inversely. One indication of how these synapses transmit information is that populations of Purkinje neurons synchronize their spikes during cerebellar behaviours. If nuclear neurons respond to Purkinje synchrony, they may encode signals from subsets of inhibitory inputs. Here we show in weanling and adult mice that nuclear neurons transmit the timing of synchronous Purkinje afferent spikes, owing to modest Purkinje-to-nuclear convergence ratios (～40:1), fast inhibitory postsynaptic current kinetics (τ(decay) = 2.5?ms) and high intrinsic firing rates (～90?Hz). In vitro, dynamically clamped asynchronous inhibitory postsynaptic potentials mimicking Purkinje afferents suppress nuclear cell spiking, whereas synchronous inhibitory postsynaptic potentials entrain nuclear cell spiking. With partial synchrony, nuclear neurons time-lock their spikes to the synchronous subpopulation of inputs, even when only 2 out of 40 afferents synchronize. In vivo, nuclear neurons reliably phase-lock to regular trains of molecular layer stimulation. Thus, cerebellar nuclear neurons can preferentially relay the spike timing of synchronized Purkinje cells to downstream premotor areas.     

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.     

Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells

Chromaffin cells release catecholamine- and peptide-containing granules by exocytosis, by a mechanism involving movement of secretory granules towards the cell membrane, their apposition to it and the fusion of the granule membrane with the plasma membrane. One of the two subunits of membrane-associated brain spectrin, alpha-fodrin is an actin-binding protein which is found at the periphery of chromaffin cells and may be involved in secretion. Because cultured chromaffin cells can be permeabilized with detergents, giving pores large enough to permit the entry of immunoglobulin molecules, we used permeabilized cells to test the effect of specific antibodies on secretory mechanisms. Incubation of permeabilized cells with polyclonal immunoaffinity-purified monospecific anti-alpha-fodrin antibody or its Fab fragments did not modify basal release but did specifically inhibit Ca2+-induced catecholamine release by exocytosis. Our observations indicate that fodrin and the cytoskeleton participate in the release mechanism.     

Horizontal cell synapses onto glycine-accumulating interplexiform cells

Horizontal cells mediate lateral transmission of signals in the outer plexiform layer of the vertebrate retina, and are presumed to contribute to surround properties of photoreceptors and bipolar cells by chemical transmission. The cell bodies and dendrites of fish horizontal cells possess presynaptic specializations characteristic of conventional chemical synapses. Horizontal cell axon terminals have not so far been shown to contain presynaptic specializations nor have the targets of the somatic and dendritic synapses been fully characterized. Using electron microscope autoradiography of retinas labelled by high-affinity 3H-glycine uptake, we show here that goldfish horizontal cells make somatodendritic and axodendritic synapses on glycinergic interplexiform cells (Gly-IPCs) as apposed to dopaminergic interplexiform cells. Thus, horizontal cells have at least three postsynaptic targets: photoreceptors, bipolar cells and Gly-IPCs. Gly-IPCs may constitute a major alternative pathway for horizontal cell signals to reach the inner plexiform layer.     

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.     

Molecular identification of a danger signal that alerts the immune system to dying cells

In infections, microbial components provide signals that alert the immune system to danger and promote the generation of immunity. In the absence of such signals, there is often no immune response or tolerance may develop. This has led to the concept that the immune system responds only to antigens perceived to be associated with a dangerous situation such as infection. Danger signals are thought to act by stimulating dendritic cells to mature so that they can present foreign antigens and stimulate T lymphocytes. Dying mammalian cells have also been found to release danger signals of unknown identity. Here we show that uric acid is a principal endogenous danger signal released from injured cells. Uric acid stimulates dendritic cell maturation and, when co-injected with antigen in vivo, significantly enhances the generation of responses from CD8+ T cells. Eliminating uric acid in vivo inhibits the immune response to antigens associated with injured cells, but not to antigens presented by activated dendritic cells. Our findings provide a molecular link between cell injury and immunity and have important implications for vaccines, autoimmunity and inflammation.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Exo1 and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells.

In many cell types an increase in cytosolic calcium is the main signal for the exocytotic release of stored secretory components such as hormones and neurotransmitters. The site of action of calcium in exocytosis is not known, neither are the participating molecules. In the case of the intracellular membrane fusions that occur during transport through early stages of the secretory pathway, several cytosolic and peripheral membrane proteins are necessary. Permeabilized cells have been useful in understanding the requirements for calcium and nucleotides in regulated exocytosis and under certain conditions there is leakage of soluble protein components and run-down of the exocytotic response. This system can be used to identify the soluble proteins involved in exocytosis, one candidate in chromaffin cells being annexin II (calpactin). Here we use this assay to identify two other cytosolic protein factors that regulate exocytosis in permeabilized adrenal chromaffin cells, which we term Exo1 and Exo2. Exo1 from brain cytosol resolves on electrophoresis in SDS-polyacrylamide gels as a group of polypeptides of relative molecular mass approximately 30,000 and shares sequence homology with the 14-3-3 family of proteins. The ability of Exo1 to reactivate exocytosis is potentiated by protein kinase C activation and therefore Exo1 may influence the protein kinase C-mediated control of Ca(2+)-dependent exocytosis.     

Presynaptic induction of heterosynaptic associative plasticity in the mammalian brain

The induction of associative synaptic plasticity in the mammalian central nervous system classically depends on coincident presynaptic and postsynaptic activity. According to this principle, associative homosynaptic long-term potentiation (LTP) of excitatory synaptic transmission can be induced only if synaptic release occurs during postsynaptic depolarization. In contrast, heterosynaptic plasticity in mammals is considered to rely on activity-independent, non-associative processes. Here we describe a novel mechanism underlying the induction of associative LTP in the lateral amygdala (LA). Simultaneous activation of converging cortical and thalamic afferents specifically induced associative, N-methyl-D-aspartate (NMDA)-receptor-dependent LTP at cortical, but not at thalamic, inputs. Surprisingly, the induction of associative LTP at cortical inputs was completely independent of postsynaptic activity, including depolarization, postsynaptic NMDA receptor activation or an increase in postsynaptic Ca2+ concentration, and did not require network activity. LTP expression was mediated by a persistent increase in the presynaptic probability of release at cortical afferents. Our study shows the presynaptic induction and expression of heterosynaptic and associative synaptic plasticity on simultaneous activity of converging afferents. Our data indicate that input specificity of associative LTP can be determined exclusively by presynaptic properties.     

Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.     

Opioids block long-term potentiation of inhibitory synapses

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.     

Observation of biophoton emission from plants in the process of defense response

Biophoton emission from germinating soybean under adverse circumstance has been observed by high sensitive imaging system based on ICCD detector. It is found that the intensity of biophoton emission from the injured position of the cotyledon is enhanced obviously compared with the intact position. In addition, a strong biophoton emission has been detected at the tip of the root when the root is supplied with water after drought. The emission induced by wounding may be associated with the defense response of plants. The emission during the process of convalescence after drought may be the results of the changes of oxidative metabolism in cells.     

Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells.

Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.     

Fast neurotransmitter release triggered by Ca influx through AMPA-type glutamate receptors

Feedback inhibition at reciprocal synapses between A17 amacrine cells and rod bipolar cells (RBCs) shapes light-evoked responses in the retina. Glutamate-mediated excitation of A17 cells elicits GABA (gamma-aminobutyric acid)-mediated inhibitory feedback onto RBCs, but the mechanisms that underlie GABA release from the dendrites of A17 cells are unknown. If, as observed at all other synapses studied, voltage-gated calcium channels (VGCCs) couple membrane depolarization to neurotransmitter release, feedforward excitatory postsynaptic potentials could spread through A17 dendrites to elicit 'surround' feedback inhibitory transmission at neighbouring synapses. Here we show, however, that GABA release from A17 cells in the rat retina does not depend on VGCCs or membrane depolarization. Instead, calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs), activated by glutamate released from RBCs, provide the calcium influx necessary to trigger GABA release from A17 cells. The AMPAR-mediated calcium signal is amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. These results describe a fast synapse that operates independently of VGCCs and membrane depolarization and reveal a previously unknown form of feedback inhibition within a neural circuit.     

NMDA receptors activate the arachidonic acid cascade system in striatal neurons

Receptors for excitatory amino-acid transmitters on nerve cells fall into two main categories associated with non-selective cationic channels, the NMDA (N-methyl-D-aspartate) and non-NMDA (kainate and quisqualate) receptors. Special properties of NMDA receptors such as their voltage-dependent blockade by Mg2+ (refs 3, 4) and their permeability to Na+, K+ as well as to Ca2+ (refs 5, 6), have led to the suggestion that these receptors are important in plasticity during development and learning. They have been implicated in long-term potentiation (LTP), a model for the study of the cellular mechanisms of learning. We report here that glutamate and NMDA, acting at typical NMDA receptors, stimulate the release of arachidonic acid (as well as 11- and 12-hydroxyeicosatetraenoic acids from striatal neurons probably by stimulation of a Ca2+-dependent phospholipase A2. Kainate and quisqualate, as well as K+-induced depolarization were ineffective. Our results provide direct evidence in favour of the hypothesis, that arachidonic acid derivatives, produced by activation of the postsynaptic cell, could be messengers that cross the synaptic cleft to modify the presynaptic functions known to be altered during LTP. In addition, we suggest that NMDA receptors are the postsynaptic receptors which trigger the synthesis of these putative transynaptic messengers.