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.     

Mitochondrial Ca2+ signalling in hippocampal neurons

High-resolution fluorescent imaging of mitochondrial-targeted probes was used to examine the ability of mitochondria to decode complex spatial and temporal Ca2+ signals evoked in synaptically active networks of hippocampal neurons. Green-to-red photoconversion of the mitochondrial-targeted probe, mito-Kaede, demonstrated that mitochondria were present as discrete organelles 2-6 microm in length. Real-time imaging of mitochondrial-targeted ratiometric pericam (2 mtRP) visualised rapid, repetitive, transient mitochondrial Ca2+ fluxes in response to periods of synaptic activation. Mitochondrial Ca2+ fluxes within cellular compartments were dependent on the extent of synaptic recruitment, but independent of cross-talk with the endoplasmic reticulum or the presence of an interconnected mitochondrial network. Mitochondria in dendritic regions demonstrated a greater sensitivity to synaptic activation compared with somatic mitochondria. Temporal decoding of synaptic signals was rate-limited by the activity of the mitochondrial Na+/Ca2+ exchanger. Spatial regulation of mitochondrial Ca2+ uptake was determined by the magnitude of the cytosolic Ca2+ rise in each cellular compartment.     

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.     

Ca2+ and synaptic plasticity

A major effort in neuroscience is directed towards understanding the roles of Ca2+ signalling in the induction of synaptic plasticity. Here, we summarize the evidence concerning Ca2+ signalling, paying particular attention to CA1 excitatory synapses, and its relationship to the induction of long-term potentiation and long-term depression. We discuss the ways in which synaptic activation can elevate Ca2+ postsynaptically and how dendritic spines may act as a Ca2+ compartment which can both isolate and integrate Ca2+ signals.     

Synchronized Ca2+ signals mediated by Ca2+ action potentials in the hippocampal neuron network in vitro

Periodic, synchronized Ca2+ signals appeared 30-120 min after the application of tetrodotoxin, 4-aminopyridine and Cs+, and became stable in interval (6-47s) for hours. The Ca2+ signals were accompanied by excitatory or inhibitory postsynaptic potentials (excitatory postsynaptic currents (EPSCs) for the former) and blocked by the simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione and 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid or treatment with Ca2+ -free solution, nicardipine, or omega-conotoxin MVIIC (omegaCTX), but not with ryanodine, caffeine, thapsigargin or CPP alone. Nicardipine largely, but omegaCTX less, blocked Ca2+ action potentials or voltage pulse-induced Ca2+ currents at the cell soma, while omegaCTX completely blocked autaptic EPSCs. Ca2+ signals within a neuron occurred almost simultaneously in the cell soma and all the processes (> 200 microm), while the latency between Ca2+ signals of neighbouring neurons varied over hundreds of ms like that of Ca2 action potential induction from EPSPs. Ca2+ signals propagated in random directions throughout neural circuits. Thus, when Na+ and K+ channels are blocked, Ca2+ action potentials spontaneously occur somewhere in a neuron, eventually propagate via the cell soma to the presynaptic terminals and activate excitatory synaptic transmission, causing synchronized Ca2+ signals. The results further suggest that the axon of hippocampal neurones have the potential ability to convey coded information via Ca2+ action potentials.     

Ca2+ and synaptic plasticity

The induction and maintenance of synaptic plasticity is well established to be a Ca2+-dependent process. The use of fluorescent imaging to monitor changes [Ca2+]i in neurones has revealed a diverse array of signaling patterns across the different compartments of the cell. The Ca2+ signals within these compartments are generated by voltage or ligand-gated Ca2+ influx, and release from intracellular stores. The changes in [Ca2+]i are directly linked to the activity of the neurone, thus a neurone's input and output is translated into a dynamic Ca2+ code. Despite considerable progress in measuring this code much still remains to be determined in order to understand how the code is interpreted by the Ca2+ sensors that underlie the induction of compartment-specific plastic changes.     

Chronic ethanol exposure enhances AMPA-elicited Ca2+ signals in the somatic and dendritic regions of cerebellar Purkinje neurons.

Intracellular Ca2+ signals produced by the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 5 microM) were measured in the somatic and dendritic regions of cerebellar Purkinje neurons in mature cerebellar control cultures (> or = 20 days in vitro) and cultures chronically treated with 32 mM ethanol (146 mg%; 8-11 days). Recordings were made in physiological saline without ethanol. The mean peak amplitude of the Ca2+ signal elicited by AMPA (applied by brief 1-s microperfusion) in the somatic region was enhanced 38% in chronic ethanol-treated Purkinje neurons compared with control neurons. In contrast, Ca2+ signals evoked by AMPA in the dendritic region were similar in magnitude between control and chronic ethanol-treated Purkinje neurons. When tetrodotoxin (TTX; 500 nM) was included in the bath saline to block spike activity and synaptically-generated events, the mean peak amplitude of the Ca2+ signal elicited by AMPA was enhanced 60% in both the somatic and dendritic regions of chronic ethanol-treated Purkinje neurons compared with control neurons. Thus, TTX-sensitive mechanisms (i.e., spike or synaptic activity) appear to play a role in normalizing neuronal functions involved in Ca2+ signaling in the chronic ethanol-treated neurons. In parallel current clamp experiments, the resting membrane potential of chronic ethanol-treated neurons was slightly depolarized compared with control neurons. However, no differences were found between control and chronic ethanol-treated Purkinje neurons in input resistance or the peak amplitude or duration of the depolarizations or hyperpolarizations elicited by AMPA. AMPA receptors mediate fast excitatory neurotransmission in the majority of neurons in the central nervous system (CNS) and Ca2+ signals in response to AMPA receptor activation contribute to synaptic function. Thus, our results suggest that modulation of Ca2+ signals to AMPA receptor activation (or other cellular inputs) may provide an important mechanism contributing to the actions of prolonged ethanol exposure in the CNS.     

Astrocyte-induced modulation of synaptic transmission

The idea that astrocytes simply provide structural and trophic support to neurons has been challenged by recent evidence demonstrating that astrocytes exhibit a form of excitability and communication based on intracellular Ca2+ variations and intercellular Ca2+ waves, which can be initiated by neuronal activity. These astrocyte Ca2+ variations have now been shown to induce glutamate-dependent Ca2+ elevations and slow inward currents in neurons. More recently, it has been demonstrated that synaptic transmission between cultured hippocampal neurons can be directly modulated by astrocytes. We have reported that astrocyte stimulation can increase the frequency of miniature synaptic currents. Furthermore, we also have demonstrated that an elevation in the intracellular Ca2+ in astrocytes induces a reduction in both excitatory and inhibitory evoked synaptic transmission through the activation of selective presynaptic metabotropic glutamate receptors.     

Role of residual calcium in synaptic depression and posttetanic potentiation: fast and slow calcium signaling in nerve terminals.

Trains of action potentials evoked rises in presynaptic Ca2+ concentration ([Ca2+]i) at the squid giant synapse. These increases in [Ca2+]i were spatially nonuniform during the trains, but rapidly equilibrated after the trains and slowly declined over hundreds of seconds. The trains also elicited synaptic depression and augmentation, both of which developed during stimulation and declined within a few seconds afterward. Microinjection of the Ca2+ buffer EGTA into presynaptic terminals had no effect on transmitter release or synaptic depression. However, EGTA injection effectively blocked both the persistent Ca2+ signals and augmentation. These results suggest that transmitter release is triggered by a large, brief, and sharply localized rise in [Ca2+]i, while augmentation is produced by a smaller, slower, and more diffuse rise in [Ca2+]i.     

Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.     

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.     

Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis

A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.     

Integrative properties of radial oblique dendrites in hippocampal CA1 pyramidal neurons

Although radial oblique dendrites are a major synaptic input site in CA1 pyramidal neurons, little is known about their integrative properties. We have used multisite two-photon glutamate uncaging to deliver different spatiotemporal input patterns to single branches while simultaneously recording the uncaging-evoked excitatory postsynaptic potentials and local Ca2+ signals. Asynchronous input patterns sum linearly in spite of the spatial clustering and produce Ca2+ signals that are mediated by NMDA receptors (NMDARs). Appropriately timed and sized input patterns ( approximately 20 inputs within approximately 6 ms) produce a supralinear summation due to the initiation of a dendritic spike. The Ca2+ signals associated with synchronous input were larger and mediated by influx through both NMDARs and voltage-gated Ca2+ channels (VGCCs). The oblique spike is a fast Na+ spike whose duration is shaped by the coincident activation of NMDAR, VGCCs, and transient K+ currents. Our results suggest that individual branches can function as single integrative compartments.     

Endogenous buffers limit the spread of free calcium in hair cells.

Mobile Ca2+ buffers in hair cells have been postulated to play a dual role. On one hand, they carry incoming Ca2+ away from synaptic areas, allowing synapses to be rapidly reset. On the other hand, they limit the spread of free Ca2+ into the cell, preventing cross-talk between different pathways that employ Ca2+ as a second messenger. We have obtained evidence for such mobile Ca2+ buffers in hair cells by comparing the patterns of Ca2+-induced fluo-3 fluorescence under whole-cell and perforated-patch recording conditions. Fluorescent signals under perforated-patch conditions are relatively weak and are limited to the immediate vicinity of the membrane. These observations can be explained by a diffusion-reaction scheme that, in addition to Ca2+ and fluo-3, incorporates endogenous fixed and mobile Ca2+ buffers. Our experiments also suggest that the mobility of the endogenous buffer might be higher than previously thought. A high buffer mobility is expected to enhance the cell's ability to rapidly modulate transmitter release.     

Inositol-1,4,5-triphosphate receptors mediate activity-induced synaptic Ca2+ signals in muscle fibers and Ca2+ overload in slow-channel syndrome

Strict control of calcium entry through excitatory synaptic receptors is important for shaping synaptic responses, gene expression, and cell survival. Disruption of this control may lead to pathological accumulation of Ca2+. The slow-channel congenital myasthenic syndrome (SCS), due to mutations in muscle acetylcholine receptor (AChR), perturbs the kinetics of synaptic currents, leading to post-synaptic Ca2+ accumulation. To understand the regulation of calcium signaling at the neuromuscular junction (NMJ) and the etiology of Ca2+ overload in SCS we studied the role of sarcoplasmic Ca2+ stores in SCS. Using fura-2 loaded dissociated fibers activated with acetylcholine puffs, we confirmed that Ca2+ accumulates around wild type NMJ and discovered that Ca2+ accumulates significantly faster around the NMJ of SCS transgenic dissociated muscle fibers. Additionally, we determined that this process is dependant on the activation, altered kinetics, and movement of Ca2+ ions through the AChR, although, surprisingly, depletion of intracellular stores also prevents the accumulation of this cation around the NMJ. Finally, we concluded that the sarcoplasmic reticulum is the main source of Ca2+ and that inositol-1,4,5-triphosphate receptors (IP3R), and to a lesser degree L-type voltage gated Ca2+ channels, are responsible for the efflux of this cation from intracellular stores. These results suggest that a signaling system mediated by the activation of AChR, Ca2+, and IP3R is responsible for localized Ca2+ signals observed in muscle fibers and the Ca2+ overload observed in SCS.     

Calcium signalling in smooth muscle

Calcium signalling in smooth muscles is complex, but our understanding of it has increased markedly in recent years. Thus, progress has been made in relating global Ca2+ signals to changes in force in smooth muscles and understanding the biochemical and molecular mechanisms involved in Ca2+ sensitization, i.e. altering the relation between Ca2+ and force. Attention is now focussed more on the role of the internal Ca2+ store, the sarcoplasmic reticulum (SR), global Ca2+ signals and control of excitability. Modern imaging techniques have shown the elaborate SR network in smooth muscles, along with the expression of IP3 and ryanodine receptors. The role and cross-talk between these two Ca(2+) release mechanisms, as well as possible compartmentalization of the SR Ca2+ store are discussed. The close proximity between SR and surface membrane has long been known but the details of this special region to Ca2+ signalling and the role of local sub-membrane Ca2+ concentrations and membrane microdomains are only now emerging. The activation of K+ and Cl- channels by local Ca2+ signals, can have profound effects on excitability and hence contraction. We examine the evidence for both Ca2+ sparks and puffs in controlling ion channel activity, as well as a fundamental role for Ca2+ sparks in governing the period of inexcitability in smooth muscle, i.e. the refractory period. Finally, the relation between different Ca2+ signals, e.g. sparks, waves and transients, to smooth muscle activity in health and disease is becoming clearer and will be discussed.     

Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria

Mitochondria in nerve terminals are subjected to extensive Ca2+ fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders.