Amplification of calcium signals at dendritic spines provides a method for CNS quantal analysis

It has been proposed that the small volume of a dendritic spine can amplify Ca2+ signals during synaptic transmission. Accordingly, we have performed calculations to determine whether the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors during synaptic transmission results in significant elevation in intracellular Ca2+ levels, permitting optical detection of synaptic signals within a single spine. Simple calculations suggest that the opening of even a single NMDA receptor would result in the influx of approximately 310 000 Ca2+ ions into the small volume of a spine, producing changes in Ca2+ levels that are readily detectable using high affinity Ca2+ indicators such as fura-2 or fluo-3. Using fluorescent Ca2+ indicators, we have imaged local Ca2+ transients mediated by NMDA receptors in spines and dendritic shafts attributed to spontaneous miniature synaptic activity. Detailed analysis of these quantal events suggests that the current triggering these transients is attributed to the activation of <10 NMDA receptors. The frequency of these miniature synaptic Ca2+ transients is not randomly distributed across synapses, as some synapses can display a >10-fold higher frequency of transients than others. As expected for events mediated by NMDA receptors, miniature synaptic Ca2+ transients were suppressed by extracellular Mg2+ at negative membrane potentials; however, the Mg2+ block could be removed by depolarization.     

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.     

Synergistic release of Ca2+ from IP3-sensitive stores evoked by synaptic activation of mGluRs paired with backpropagating action potentials

Increases in postsynaptic [Ca2+]i can result from Ca2+ entry through ligand-gated channels or voltage-gated Ca2+ channels, or through release from intracellular stores. Most attention has focused on entry through the N-methyl-D-aspartate (NMDA) receptor in causing [Ca2+]i increases since this pathway requires both presynaptic stimulation and postsynaptic depolarization, making it a central component in models of synaptic plasticity. Here, we report that repetitive synaptic activation of metabotropic glutamate receptors (mGluRs), paired with backpropagating action potentials, causes large, wave-like increases in [Ca2+]i predominantly in restricted regions of the proximal apical dendrites and soma of hippocampal CA1 pyramidal neurons. [Ca2+]i changes of several micromolars can be reached by regenerative release caused by the synergistic effect of mGluR-generated inositol 1,4,5-trisphosphate (IP3) and spike-evoked Ca2+ entry acting on the IP3 receptor.     

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.     

Local learning rules: predicted influence of dendritic location on synaptic modification in spike-timing-dependent plasticity

Recent indirect experimental evidence suggests that synaptic plasticity changes along the dendrites of a neuron. Here we present a synaptic plasticity rule which is controlled by the properties of the pre- and postsynaptic signals. Using recorded membrane traces of back-propagating and dendritic spikes we demonstrate that LTP and LTD will depend specifically on the shape of the postsynaptic depolarization at a given dendritic site. We find that asymmetrical spike-timing-dependent plasticity (STDP) can be replaced by temporally symmetrical plasticity within physiologically relevant time windows if the postsynaptic depolarization rises shallow. Presynaptically the rule depends on the NMDA channel characteristic, and the model predicts that an increase in Mg²⁺ will attenuate the STDP curve without changing its shape. Furthermore, the model suggests that the profile of LTD should be governed by the postsynaptic signal while that of LTP mainly depends on the presynaptic signal shape.     

Bidirectional plasticity gated by hyperpolarization controls the gain of postsynaptic firing responses at central vestibular nerve synapses

Linking synaptic plasticity with behavioral learning requires understanding how synaptic efficacy influences postsynaptic firing in neurons whose role in behavior is understood. Here, we examine plasticity at a candidate site of motor learning: vestibular nerve synapses onto neurons that mediate reflexive movements. Pairing nerve activity with changes in postsynaptic voltage induced bidirectional synaptic plasticity in vestibular nucleus projection neurons: long-term potentiation relied on calcium-permeable AMPA receptors and postsynaptic hyperpolarization, whereas long-term depression relied on NMDA receptors and postsynaptic depolarization. Remarkably, both forms of plasticity uniformly scaled synaptic currents evoked by pulse trains, and these changes in synaptic efficacy were translated into linear increases or decreases in postsynaptic firing responses. Synapses onto local inhibitory neurons were also plastic but expressed only long-term depression. Bidirectional, linear gain control of vestibular nerve synapses onto projection neurons provides a plausible mechanism for motor learning underlying adaptation of vestibular reflexes.     

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.     

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).     

Novel presynaptic mechanisms for coincidence detection in synaptic plasticity

Long-term plasticity typically relies on postsynaptic NMDA receptors to detect the coincidence of pre- and postsynaptic activity. Recent studies, however, have revealed forms of plasticity that depend on coincidence detection by presynaptic NMDA receptors. In the amygdala, cortical afferent associative presynaptic long-term potentiation (LTP) requires activation of presynaptic NMDA receptors by simultaneous thalamic and cortical afferents. Surprisingly, both types of afferent can also undergo postsynaptically induced NMDA-receptor-dependent LTP. In the neocortex, spike-timing-dependent long-term depression (LTD) requires simultaneous activation of presynaptic NMDA autoreceptors and retrograde signalling by endocannabinoids. In cerebellar LTD, presynaptic NMDA receptor activation suggests that similar presynaptic mechanisms may exist. Recent studies also indicate the existence of presynaptic coincidence detection that is independent of NMDA receptors, suggesting that such mechanisms have a widespread role in plasticity.     

GABAB receptor modulation of synaptic function

Neuromodulators have complex effects on both the presynaptic release and postsynaptic detection of neurotransmitters. Here we describe recent advances in our understanding of synaptic modulation by metabotropic GABAB receptors. By inhibiting multivesicular release from the presynaptic terminal, these receptors decrease the synaptic glutamate signal. GABAB receptors also inhibit the Ca2+ permeability of NMDA receptors to decrease Ca2+ signals in postsynaptic spines. These new findings highlight the importance of GABAB receptors in regulating many aspects of synaptic transmission. They also point to novel questions about the spatiotemporal dynamics and sources of synaptic modulation in the brain.     

PTEN is recruited to the postsynaptic terminal for NMDA receptor‐dependent long‐term depression

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important regulator of phosphatidylinositol‐(3,4,5,)‐trisphosphate signalling, which controls cell growth and differentiation. However, PTEN is also highly expressed in the adult brain, in which it can be found in dendritic spines in hippocampus and other brain regions. Here, we have investigated specific functions of PTEN in the regulation of synaptic function in excitatory hippocampal synapses. We found that NMDA receptor activation triggers a PDZ‐dependent association between PTEN and the synaptic scaffolding molecule PSD‐95. This association is accompanied by PTEN localization at the postsynaptic density and anchoring within the spine. On the other hand, enhancement of PTEN lipid phosphatase activity is able to drive depression of AMPA receptor‐mediated synaptic responses. This activity is specifically required for NMDA receptor‐dependent long‐term depression (LTD), but not for LTP or metabotropic glutamate receptor‐dependent LTD. Therefore, these results reveal PTEN as a regulated signalling molecule at the synapse, which is recruited to the postsynaptic membrane upon NMDA receptor activation, and is required for the modulation of synaptic activity during plasticity.     

A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons.

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Determinants of postsynaptic Ca2+ signaling in Purkinje neurons

Neuronal integration in Purkinje neurons involves many forms of Ca2+ signaling. Two afferent synaptic inputs, the parallel and the climbing fibers, provide a major drive for these signals. These two excitatory synaptic inputs are both glutamatergic. Postsynaptically they activate alpha-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Unlike most other types of central neurons, Purkinje neurons do not express NMDA (N-methyl-D-aspartate) receptors (NMDARs). AMPARs in Purkinje neurons are characterized by a low permeability for Ca2+ ions. AMPAR-mediated synaptic depolarization may activate voltage-gated Ca2+ channels, mostly of the P/Q-type. The resulting intracellular Ca2+ signals are shaped by the Ca2+ buffers calbindin and parvalbumin. Ca2+ clearance from the cytosol is brought about by Ca2+-ATPases in the plasma membrane and the endoplasmic reticulum, as well as the Na+-Ca2+-exchanger. Binding of glutamate to mGluRs induces postsynaptic Ca2+-transients through two G protein-dependent pathways: involving (1) the release of Ca2+ ions from intracellular Ca2+ stores and (2) the opening of the cation channel TRPC1. Homer proteins appear to play an important role in postsynaptic Ca2+ signaling by providing a direct link between the plasma membrane-resident elements (mGluRs and TRPC1) and their intracellular partners, including the IP3Rs.     

Common molecular pathways mediate long-term potentiation of synaptic excitation and slow synaptic inhibition

Synaptic plasticity, the cellular correlate for learning and memory, involves signaling cascades in the dendritic spine. Extensive studies have shown that long-term potentiation (LTP) of the excitatory postsynaptic current (EPSC) through glutamate receptors is induced by activation of N-methyl-D-asparate receptor (NMDA-R)--the coincidence detector--and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Here we report that the same signaling pathway in the postsynaptic CA1 pyramidal neuron also causes LTP of the slow inhibitory postsynaptic current (sIPSC) mediated by metabotropic GABA(B) receptors (GABA(B)-Rs) and G protein-activated inwardly rectifying K(+) (GIRK) channels, both residing in dendritic spines as well as shafts. Indicative of intriguing differences in the regulatory mechanisms for excitatory and inhibitory synaptic plasticity, LTP of sIPSC but not EPSC was abolished in mice lacking Nova-2, a neuronal-specific RNA binding protein that is an autoimmune target in paraneoplastic opsoclonus myoclonus ataxia (POMA) patients with latent cancer, reduced inhibitory control of movements, and dementia.     

Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses.

The protein brain-derived neurotrophic factor (BDNF) has been postulated to be a retrograde or paracrine synaptic messenger in long-term potentiation and other forms of activity-dependent synaptic plasticity. Although crucial for this concept, direct evidence for the activity-dependent synaptic release of BDNF is lacking. Here we investigate secretion of BDNF labelled with green fluorescent protein (BDNF-GFP) by monitoring the changes in fluorescence intensity of dendritic BDNF-GFP vesicles at glutamatergic synaptic junctions of living hippocampal neurons. We show that high-frequency activation of glutamatergic synapses triggers the release of BDNF-GFP from synaptically localized secretory granules. This release depends on activation of postsynaptic ionotropic glutamate receptors and on postsynaptic Ca(2+) influx. Release of BDNF-GFP is also observed from extrasynaptic dendritic vesicle clusters, suggesting that a possible spatial restriction of BDNF release to specific synaptic sites can only occur if the postsynaptic depolarization remains local. These results support the concept of BDNF being a synaptic messenger of activity-dependent synaptic plasticity, which is released from postsynaptic neurons.     

Mechanisms and significance of spike-timing dependent plasticity

Hebb's original postulate left two important issues unaddressed: (i) what is the effective time window between pre- and postsynaptic activity that will result in potentiation? and (ii) what is the learning rule that underlies decreases in synaptic strength? While research over the past 2 decades has addressed these questions, several studies within the past 5 years have shown that synapses undergo long-term depression (LTD) or long-term potentiation (LTP) depending on the order of activity in the pre- and postsynaptic cells. This process has been referred to as spike-timing dependent plasticity (STDP). Here we discuss the experimental data on STDP, and develop models of the mechanisms that may underlie it. Specifically, we examine whether the standard model of LTP and LTD in which high and low levels of Ca(2+) produce LTP and LTD, respectively, can also account for STDP. We conclude that the standard model can account for a type of STDP in which, counterintuitively, LTD will be observed at some intervals in which the presynaptic cell fires before the postsynaptic cell. This form of STDP will also be sensitive to parameters such as the presence of an after depolarization following an action potential. Indeed, the sensitivity of this type of STDP to experimental parameters suggests that it may not play an important physiological role in vivo. We suggest that more robust forms of STDP, which do not exhibit LTD at pre-before-post intervals, are not accounted for by the standard model, and are likely to rely on a second coincidence detector in addition to the NMDA receptor.     

Participation of NMDA-mediated phosphorylation and oxidation of neurogranin in the regulation of Ca2+- and Ca2+/calmodulin-dependent neuronal signaling in the hippocampus

Neurogranin/RC3 (Ng) is a postsynaptic protein kinase C (PKC) substrate and calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by Ca2+, phosphorylation and oxidation. Ng has been implicated in the modulation of postsynaptic signal transduction pathways and synaptic plasticity. Previously, we showed a severe deficit of spatial memory in Ng knockout (KO) mice. Activation of the NMDA receptor and its downstream signaling molecules are known to be involved in long-term memory formation. In the present study, using mouse hippocampal slices, we demonstrated that NMDA induced a rapid and transient phosphorylation and oxidation of Ng. NMDA also caused activation of PKC as evidenced by their phosphorylations, whereas, such activations were greatly reduced in the KO mice. A higher degree of phosphorylation of Ca2+/CaM-dependent kinase II and activation of cyclic AMP-dependent protein kinase were also evident in the WT compared to those of the KO mice. Phosphorylation of downstream targets, including mitogen-activated protein kinases and cAMP response element-binding protein, were significantly attenuated in the KO mice. These results suggest that by its Ca2+-sensitive CaM-binding feature, and through its phosphorylation and oxidation, Ng regulates the Ca2+- and Ca2+/CaM-dependent signaling pathways subsequent to the stimulation of NMDA receptor. These findings support the hypothesis that the derangement of hippocampal signal transduction cascades in Ng KO mice causes the deficits in synaptic plasticity, learning and memory that occur in these mice.     

Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.     

Displacement of alpha-actinin from the NMDA receptor NR1 C0 domain By Ca2+/calmodulin promotes CaMKII binding

Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and alpha-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing alpha-actinin from NR1 C0 upon Ca2+ influx. We show that Ca2+-depleted calmodulin and alpha-actinin simultaneously bind to NR1 C0. Upon addition of Ca2+, calmodulin dislodges alpha-actinin. Either the N- or C-terminal half of calmodulin is sufficient for Ca2+-induced displacement of alpha-actinin. Whereas alpha-actinin directly antagonizes CaMKII binding to NR1 C0, the addition of Ca2+/calmodulin shifts binding of NR1 C0 toward CaMKII by displacing alpha-actinin. Displacement of alpha-actinin results in the simultaneous binding of calmodulin and CaMKII to NR1 C0. Our results reveal an intricate mechanism whereby Ca2+ functions to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII.