Calcium-Dependent Desensitization of NMDA Receptors

Glutamate receptors play the key role in excitatory synaptic transmission in the central nervous system (CNS). N-methyl-D-aspartate-activated glutamate receptors (NMDARs) are ion channels permeable to sodium, potassium, and calcium ions that localize to the pre- and postsynaptic membranes, as well as extrasynaptic neuronal membrane. Calcium entry into dendritic spines is essential for long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission. Both LTP and LTD represent morphological and functional changes occurring in the process of memory formation. NMDAR dysfunction is associated with epilepsy, schizophrenia, migraine, dementia, and neurodegenerative diseases. Prolonged activation of extrasynaptic NMDARs causes calcium overload and apoptosis of neurons. Here, we review recent findings on the molecular mechanisms of calcium-dependent NMDAR desensitization that ensures fast modulation of NMDAR conductance in the CNS and limits calcium entry into the cells under pathological conditions. We present the data on molecular determinants related to calcium-dependent NMDAR desensitization and functional interaction of NMDARs with other ion channels and transporters. We also describe association of NMDARs with lipid membrane microdomains.     

Computational investigation of the changing patterns of subtype specific NMDA receptor activation during physiological glutamatergic neurotransmission

NMDA receptors (NMDARs) are the major mediator of the postsynaptic response during synaptic neurotransmission. The diversity of roles for NMDARs in influencing synaptic plasticity and neuronal survival is often linked to selective activation of multiple NMDAR subtypes (NR1/NR2A-NMDARs, NR1/NR2B-NMDARs, and triheteromeric NR1/NR2A/NR2B-NMDARs). However, the lack of available pharmacological tools to block specific NMDAR populations leads to debates on the potential role for each NMDAR subtype in physiological signaling, including different models of synaptic plasticity. Here, we developed a computational model of glutamatergic signaling at a prototypical dendritic spine to examine the patterns of NMDAR subtype activation at temporal and spatial resolutions that are difficult to obtain experimentally. We demonstrate that NMDAR subtypes have different dynamic ranges of activation, with NR1/NR2A-NMDAR activation sensitive at univesicular glutamate release conditions, and NR2B containing NMDARs contributing at conditions of multivesicular release. We further show that NR1/NR2A-NMDAR signaling dominates in conditions simulating long-term depression (LTD), while the contribution of NR2B containing NMDAR significantly increases for stimulation frequencies that approximate long-term potentiation (LTP). Finally, we show that NR1/NR2A-NMDAR content significantly enhances response magnitude and fidelity at single synapses during chemical LTP and spike timed dependent plasticity induction, pointing out an important developmental switch in synaptic maturation. Together, our model suggests that NMDAR subtypes are differentially activated during different types of physiological glutamatergic signaling, enhancing the ability for individual spines to produce unique responses to these different inputs.     

Validation of organotypical hippocampal slice cultures as an ex vivo model of brain ischemia: different roles of NMDA receptors in cell death signalling after exposure to NMDA or oxygen and glucose deprivation

N-Methyl-D-aspartate receptors (NMDARs) are essential mediators of synaptic plasticity under normal physiological conditions. During brain ischemia, these receptors are excessively activated due to glutamate overflow and mediate excitotoxic cell death. Although organotypical hippocampal slice cultures are widely used to study brain ischemia in vitro by induction of oxygen and glucose deprivation (OGD), there is scant data regarding expression and functionality of NMDARs in such slice cultures. Here, we have evaluated the contribution of NMDARs in mediating excitotoxic cell death after exposure to NMDA or OGD in organotypical hippocampal slice cultures after 14 days in vitro (DIV14). We found that all NMDAR subunits were expressed at DIV14. The NMDARs were functional and contributed to cell death, as evidenced by use of the NMDAR antagonist MK-801 (dizocilpine). Excitotoxic cell death induced by NMDA could be fully antagonized by 10 μM MK-801, a dose that offered only partial protection against OGD-induced cell death. Very high concentrations of MK-801 (50–100 μM) were required to counteract cell death at long delays (48–72 h) after OGD. The relative high dose of MK-801 needed for long-term protection after OGD could not be attributed to down-regulation of NMDARs at the gene expression level. Our data indicate that NMDAR signaling is just one of several mechanisms underlying ischemic cell death and that prospective cytoprotective therapies must be directed to multiple targets.     

Tyrosine dephosphorylation and ethanol inhibition of N-Methyl-D-aspartate receptor function

The inhibitory effect of ethanol on N-methyl-d-aspartate receptors (NMDARs) is well documented in several brain regions. However, the molecular mechanisms by which ethanol affects NMDARs are not well understood. In contrast to the inhibitory effect of ethanol, phosphorylation of the NMDAR potentiates channel currents (Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci. 2, 331-338). We have previously shown that protein kinase C activators induce tyrosine phosphorylation and potentiation of the NMDAR (Grosshans, D. R., Clayton, D. R., Coultrap, S. J., and Browning, M. D. (2002) Nat. Neurosci. 5, 27-33). We therefore hypothesized that the ethanol inhibition of NMDARs might be due to changes in tyrosine phosphorylation of NMDAR subunits. In support of this hypothesis, we found that tyrosine phosphorylation of both NR2A and NR2B subunits was significantly reduced following in situ exposure of hippocampal slices to 100 mm ethanol. Specifically, phosphorylation of tyrosine 1472 on NR2B was reduced 23.5%. These data suggest a possible mechanism by which ethanol may inhibit the NMDAR via activation of a tyrosine phosphatase. Electrophysiological studies demonstrated that ethanol inhibited NMDAR field excitatory postsynaptic potential slope and amplitude to a similar degree as previously reported by our laboratory and others (Schummers, J., Bentz, S., and Browning, M. D. (1997) Alcohol Clin. Exp. Res. 21, 404-408). Inclusion of bpV(phen), a potent phosphotyrosine phosphatase inhibitor, in the recording chamber prior to and during ethanol exposure significantly reduced the inhibitory effect of ethanol on NMDAR field excitatory postsynaptic potentials. Taken together, these data suggest that phosphatase-mediated dephosphorylation of NMDAR subunits may play an important role in mediating the inhibitory effects of ethanol on the N-methyl-D-aspartate receptor.     

NMDA receptor subunits: diversity, development and disease

N-methyl-D-aspartate receptors (NMDARs) are present at many excitatory glutamate synapses in the central nervous system and display unique properties that depend on their subunit composition. Biophysical, pharmacological and molecular methods have been used to determine the key features conferred by the various NMDAR subunits, and have helped to establish which NMDAR subtypes are present at particular synapses. Recent studies are beginning to address the functional significance of NMDAR diversity under normal and pathological conditions.     

Marked attenuation of inflammatory mediator-induced C-fiber sensitization for mechanical and hypotonic stimuli in TRPV4-/- mice

NMDA receptors (NMDARs) are involved in excitatory synaptic transmission and plasticity associated with a variety of brain functions, from memory formation to chronic pain. Subunit-selective antagonists for NMDARs provide powerful tools to dissect NMDAR functions in neuronal activities. Recently developed antagonist for NR2A-containing receptors, NVP-AAM007, triggered debates on its selectivity and involvement of the NMDAR subunits in bi-directional synaptic plasticity. Here, we re-examined the pharmacological properties of NMDARs in the anterior cingulate cortex (ACC) using NVP-AAM007 as well as ifenprodil, a selective antagonist for NR2B-containing NMDARs. By alternating sequence of drug application and examining different concentrations of NVP-AAM007, we found that the presence of NVP-AAM007 did not significantly affect the effect of ifenprodil on NMDAR-mediated EPSCs. These results suggest that NVP-AAM007 shows great preference for NR2A subunit and could be used as a selective antagonist for NR2A-containing NMDARs in the ACC.     

Do specific NMDA receptor subunits act as gateways for addictive behaviors?

Addiction to alcohol and drugs is a major social and economic problem, and there is considerable interest in understanding the molecular mechanisms that promote addictive drives. A number of proteins have been identified that contribute to expression of addictive behaviors. NMDA receptors (NMDARs), a subclass of ionotropic glutamate receptors, have been of particular interest because their physiological properties make them an attractive candidate for gating induction of synaptic plasticity, a molecular change thought to mediate learning and memory. NMDARs are generally inactive at the hyperpolarized resting potentials of many neurons. However, given sufficient depolarization, NMDARs are activated and exhibit long‐lasting currents with significant calcium permeability. Also, in addition to stimulating neurons by direct depolarization, NMDARs and their calcium signaling can allow strong and/or synchronized inputs to produce long‐term changes in other molecules (such as AMPA‐type glutamate receptors) which can last from days to years, binding internal and external stimuli in a long‐term memory trace. Such memories could allow salient drug‐related stimuli to exert strong control over future behaviors and thus promote addictive drives. Finally, NMDARs may themselves undergo plasticity, which can alter subsequent neuronal stimulation and/or the ability to induce plasticity. This review will address recent and past findings suggesting that NMDAR activity promotes drug‐ and alcohol‐related behaviors, with a particular focus on GluN2B subunits as possible central regulators of many addictive behaviors, as well as newer studies examining the importance of non‐canonical NMDAR subunits and endogenous NMDAR cofactors.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.     

A Role for Calcium-Permeable AMPA Receptors in Synaptic Plasticity and Learning

A central concept in the field of learning and memory is that NMDARs are essential for synaptic plasticity and memory formation. Surprisingly then, multiple studies have found that behavioral experience can reduce or eliminate the contribution of these receptors to learning. The cellular mechanisms that mediate learning in the absence of NMDAR activation are currently unknown. To address this issue, we examined the contribution of Ca²⁺-permeable AMPARs to learning and plasticity in the hippocampus. Mutant mice were engineered with a conditional genetic deletion of GluR2 in the CA1 region of the hippocampus (GluR2-cKO mice). Electrophysiology experiments in these animals revealed a novel form of long-term potentiation (LTP) that was independent of NMDARs and mediated by GluR2-lacking Ca²⁺-permeable AMPARs. Behavioral analyses found that GluR2-cKO mice were impaired on multiple hippocampus-dependent learning tasks that required NMDAR activation. This suggests that AMPAR-mediated LTP interferes with NMDAR-dependent plasticity. In contrast, NMDAR-independent learning was normal in knockout mice and required the activation of Ca²⁺-permeable AMPARs. These results suggest that GluR2-lacking AMPARs play a functional and previously unidentified role in learning; they appear to mediate changes in synaptic strength that occur after plasticity has been established by NMDARs.     

mGluR5 and NMDA receptors drive the experience- and activity-dependent NMDA receptor NR2B to NR2A subunit switch

In cerebral cortex there is a developmental switch from NR2B- to NR2A-containing NMDA receptors (NMDARs) driven by activity and sensory experience. This subunit switch alters NMDAR function, influences synaptic plasticity, and its dysregulation is associated with neurological disorders. However, the mechanisms driving the subunit switch are not known. Here, we show in hippocampal CA1 pyramidal neurons that the NR2B to NR2A switch driven acutely by activity requires activation of NMDARs and mGluR5, involves PLC, Ca(2+) release from IP(3)R-dependent stores, and PKC activity. In mGluR5 knockout mice the developmental NR2B-NR2A switch in CA1 is deficient. Moreover, in visual cortex of mGluR5 knockout mice, the NR2B-NR2A switch evoked in?vivo by visual experience is absent. Thus, we establish that mGluR5 and NMDARs are required for the activity-dependent NR2B-NR2A switch and play a critical role in experience-dependent regulation of NMDAR subunit composition in?vivo.     

Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia

The N-methyl-D-aspartate (NMDA) type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 microM NMDA or glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (< or = 10 microM) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h of stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of autoregulation of the receptor probably operates during cerebral ischemia, because NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Because NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning.     

Synaptic and Extrasynaptic NMDA Receptors Are Gated by Different Endogenous Coagonists

N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in?a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.     

Withdrawal from Cocaine Self-Administration Alters NMDA Receptor-Mediated Ca(2+) Entry in Nucleus Accumbens Dendritic Spines

We previously showed that the time-dependent intensification ("incubation") of cue-induced cocaine seeking after withdrawal from extended-access cocaine self-administration is accompanied by accumulation of Ca(2+)-permeable AMPA receptors (CP-AMPARs) in the rat nucleus accumbens (NAc). These results suggest an enduring change in Ca(2+) signaling in NAc dendritic spines. The purpose of the present study was to determine if Ca(2+) signaling via NMDA receptors (NMDARs) is also altered after incubation. Rats self-administered cocaine or saline for 10 days (6 h/day). After 45-47 days of withdrawal, NMDAR-mediated Ca(2+) entry elicited by glutamate uncaging was monitored in individual NAc dendritic spines. NMDAR currents were simultaneously recorded using whole cell patch clamp recordings. We also measured NMDAR subunit levels in a postsynaptic density (PSD) fraction prepared from the NAc of identically treated rats. NMDAR currents did not differ between groups, but a smaller percentage of spines in the cocaine group responded to glutamate uncaging with NMDAR-mediated Ca(2+) entry. No significant group differences in NMDAR subunit protein levels were found. The decrease in the proportion of spines showing NMDAR-mediated Ca(2+) entry suggests that NAc neurons in the cocaine group contain more spines which lack NMDARs (non-responding spines). The fact that cocaine and saline groups did not differ in NMDAR currents or NMDAR subunit levels suggests that the number of NMDARs on responding spines is not significantly altered by cocaine exposure. These findings are discussed in light of increases in dendritic spine density in the NAc observed after withdrawal from repeated cocaine exposure.     

D1 and D4 dopaminergic receptor interplay mediates coincident G protein-independent and dependent regulation of glutamate NMDA receptors in the lateral amygdala

Dopamine (DA) receptor and NMDA receptor (NMDAR) activation in the lateral (LA) nucleus of the amygdala plays a critical role in emotional processing. Several distinct mechanisms regulate the molecular cross-talk between DA receptors and NMDARs in different brain regions; however, the cellular mechanism through which DA modulates NMDARs in LA projection neurons has not been studied. Here, we investigated the effect of DA receptor activation on NMDAR currents in LA projection neurons recorded in amygdala slices obtained from young rats. We found that DA reduces NMDAR current amplitudes in an additive manner through the activation of both D1-like and D2-like receptors. The reduction of NMDAR current amplitudes by D1-like receptor activation is mediated by a protein-protein interaction between the D1R and the NMDAR, while the regulation of NMDAR activity by D2-like receptors is elicited through a G protein-dependent pathway controlled by D4R. The results of our investigation show for the first time a functional interplay between D1R and D4R that mediates coincident G protein-independent and dependent regulation of NMDARs.     

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.     

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95

Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS-PSD-95 interaction via overexpressing the N-terminal amino acid residues 1-133 of nNOS (nNOS-N(1-133)) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS-PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.     

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.     

Dendritic NMDA receptors activate axonal calcium channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.